Method for introducing sirna into cells by photochemical internalisation

ABSTRACT

The present invention relates to a method for introducing an siRNA molecule into the cytosol of a cell, said method comprising i) contacting said cell with an siRNA molecule, a carrier and a photosensiting agent, and ii) irradiating the cell with light of a wavelength effective to activate the photosensitising agent, wherein said carrier comprises a cationic polyamine such as a lipopolyamine in a non-liposomal formulation, polyethyleneimine (PEI), a betacyclodextrin amine polymer, an amine group containing dendrimer, and a cationic peptide. Cells or a population of cells obtainable by the method, a composition containing an siRNA molecule and the carrier molecule, kits and therapeutic uses of the above are also provided.

The present invention relates to a method for introducing shortinterfering RNA (siRNA) into cells, preferably into the cytosol ofcells, using a photosensitising agent and a carrier molecule andirradiation of the cells with light of a wavelength effective toactivate the photosensitising agent, and to the use of this method foraltering gene activity, e.g. gene silencing in vitro or in vivo.

The process of RNA interference occurs in many organisms and in thisprocess, double-stranded non-coding RNA silences gene expression in asequence specific, post-transcriptional manner. In nature, thephenomenon protects an organism's genome from foreign, invading nucleicacids such as transposons, transgenes and viral genes.

The introduction of double-stranded RNA (dsRNA) into a cell triggersthis process of RNA silencing, and any mRNA in the cell corresponding insequence to the introduced dsRNA is degraded. RNA silencing pathwaysinvolve the conversion of dsRNA into short interfering RNAs (siRNAs)that direct ribonucleases to homologous mRNA targets. The enzyme Dicerprocesses the dsRNA into siRNAs, which are in general 20-25 nucleotideslong. The siRNAs then assemble into endoribonuclease-containingcomplexes known as RNA-induced silencing complexes (RISCs), which areguided to complementary RNA molecules, where they cleave and destroy thetarget mRNA. Small amounts of dsRNA can silence a large amount of targetmRNA due to an amplification component of RNA silencing (reviewed inHannon and Rossi (2004), Nature 431, 371-378).

Knowledge that siRNA molecules are key components of the pathway led tothe testing of chemically synthesised siRNA molecules of approximately20 to 22 base pairs in length, corresponding to targeted RNA or DNAsequences. These molecules were shown to act to disrupt the expressionof the targeted sequences in mammalian cells (Elbashir S. M. et al.,(2001) Nature 411, 494-498). A 20 nucleotide siRNA is usuallysufficiently long to induce gene-specific silencing, but sufficientlyshort to evade a host response. The decrease in expression of targetedgene products can be extensive with 90% silencing induced by a fewmolecules of siRNA.

siRNA technology has thus been developed as a general technique forsequence specific gene silencing. Gene silencing has many applications,both in vitro and in vivo, as both a research tool and a therapeuticstrategy. The high potency and specificity that is seen when siRNAtechnology is used makes this technology particularly attractive.

In all cases, delivery of the siRNA molecules to the cells represents amajor challenge, as for gene silencing to occur, it is necessary for thesiRNA molecules to enter the cells in sufficient concentrations to beuseful. The strength of the silencing response and its duration isaffected by the amount of siRNA that is delivered to the cell, and ithas been shown that by supplying the siRNA in sufficiently highconcentrations, even a relatively weak siRNA molecule can silence itstarget. This should however be balanced against the fact that it isknown that administering large amounts of siRNA into a cell can lead toundesired effects such as “off target” effects (i.e. unwanted changes inprotein expression levels) or the activation of innate immune pathways.

In general, siRNA has been applied to cells by using standardtransfection protocols for nucleic acids, such as by using liposomes,cationic lipids, anionic lipids, and microinjection. siRNA is a doublestranded molecule and as such, delivery and cellular uptake of themolecule is more difficult than for antisense, which binds to serumproteins to be taken up by cells. Various different strategies have beenused, and commercially available kits exist for this purpose. As notedabove, efficient transfection is highly desirable since the potency ofgene silencing is at least in part dependent on the concentration ofsiRNA in the cell, but administration to cells in high concentrationsmay also cause undesirable side effects.

Administration at high levels also often requires high concentrations oftransfection reagents, and this can have adverse effects on the cellsincluding reduced cell viability and various other side effects, bothphenotypic and non-phenotypic. Furthermore, when high concentrations ofreagents are used, specific delivery is not achieved.

Targeted delivery of nucleic acid molecules such as siRNA is also, ingeneral, not reliable enough. Viruses could be used for this purpose,however, there are safety concerns with this approach and systemic viraldelivery is difficult to achieve.

siRNA acts in the cytosol of cells, and it is necessary for the moleculeto reach the cytosol for it to act. In view of the above considerations,it would be desirable to develop an improved method of delivering siRNAto the cytosol of a cell. Desirable properties of such an improvedmethod include i) the ability to generate time and site specificdelivery of siRNA molecules to its site of action, ii) the avoidance ofthe use of high concentrations of transfection reagent and/or siRNAand/or iii) enhanced siRNA silencing in cell lines. In particular, suchmethods would reduce the overall number of siRNA:lipid complexesrequired to achieve a certain level of gene silencing or improve on it.In such methods the siRNA:transfection reagent ratio may be alteredwhilst maintaining a certain amount of gene silencing or improving onit. Increasing the siRNA:lipid ratio is useful since it would minimizethe inhibitor effects that are observed when using high concentrationsof transfection reagents.

The overall aim of the improved method can alternatively be stated as adesire to balance the need for effective and controllable siRNA deliveryto the cytosol with reduction of adverse side effects caused by eitherhigh concentrations of transfection reagents, or non-specific effects,e.g. in particular cell types. As noted above, the reduction of theoverall number of siRNA:lipid complexes and/or an increase in thesiRNA:lipid ratio would contribute to this goal.

In order to achieve these goals, the inventors have combined the use ofa carrier (transfection reagent) with the technique of photochemicalinternalisation (PCI). The particular carrier that is chosen deliversthe siRNA molecule to intracellular compartments of the cell, e.g.endocytic vesicles such as the endosome and/or the lysosome of the cell.Alternative intracellular compartments into which the siRNA:lipidcomplex may be taken up include the Golgi apparatus and the endoplasmicreticulum.

Release of the siRNA molecule from the intracellular vesicle occurs as aconsequence of the PCI technique. This is dependent on exposure of thecell to a photosensitising compound and subsequent irradiation, and itcan be seen that the release of the siRNA molecule only occurs followingirradiation of the cell and as such, this release into the cytosol whereits effects are mediated can be controlled in a spatial or temporalmanner. Only cells which i) contain siRNA in their intracellularvesicles, ii) have been exposed to the photochemical internaliser andiii) are exposed to irradiation, will release the siRNA molecule intothe cytosol of the cell for it to act on mRNA in that cell.

In general, transfection reagents need to be used at high concentrationsto optimise the delivery of siRNA to the cytosol. The inventors havesurprisingly observed that by using low concentrations of transfectionreagents (and a photochemical internaliser), the transfection step canbe used to direct the siRNA to intracellular vesicles, such as theendosome, where it is contained until its release is triggered by theapplication of irradiation. The method thus allows siRNA to reach itssite of action without the need to use high concentrations oftransfection reagent or siRNA. Furthermore, the timing and location ofthe release of the siRNA molecule from intracellular vesicles such asthe endosome can be controlled by using the PCI technique.

Thus, in a first aspect, the invention provides a method for introducingan siRNA molecule into the cytosol of a cell, comprising contacting saidcell with an siRNA molecule, a carrier and a photosensitising agent, andirradiating the cell with light of a wavelength effective to activatethe photosensitising agent. Once activated, intracellular compartmentswithin said cell containing said photosensitising agent release siRNAcontained in these compartments into the cytosol.

PCI is a technique which uses a photosensitising agent, in combinationwith an irradiation step to activate that agent, and achieves release ofmolecules co-administered to the cell into the cell's cytosol. Thistechnique allows molecules that are taken up by the cell intoorganelles, such as endosomes, to be released from these organelles intothe cytosol, following irradiation.

The basic method of photochemical internalisation (PCI), is described inWO 96/07432 and WO 00/54802, which are incorporated herein by reference.As set out above, the molecule to be internalised (which for useaccording to the present invention would be the siRNA molecule), in thiscase with a carrier molecule, and a photosensitising agent are broughtinto contact with a cell. The photosensitising agent, carrier moleculeand the molecule to be internalised are taken up into a cellularmembrane-bound subcompartment within the cell. On exposure of the cellto light of the appropriate wavelength, the photosensitizing agent isactivated which directly or indirectly generates reactive species whichdisrupt the intracellular compartment membranes. This allows theinternalized molecule to be released into the cytosol.

These methods use the photochemical effect as a mechanism forintroducing otherwise membrane-impermeable (or poorly permeable)molecules into the cytosol of a cell in a manner which does not resultin widespread cell destruction or cell death if the methodology issuitably adjusted to avoid excessive toxic species production, e.g. bylowering illumination times or photosensitizer dose.

This method is particularly advantageous for introducing siRNA intocells because it allows the use of lower concentrations of carrier ortransfection reagent and/or siRNA than is required for conventionalsiRNA transfection, whilst achieving effective gene inhibition.Furthermore, the timing and location of irradiation to release the siRNAmolecule may be controlled such that it is released only at the time andlocation that is desired to achieve the required effects. As such,exposure of cells to siRNA and carrier is minimised, and undesirableside effects are minimised. This is in contrast to the standardtechniques for introducing siRNA into cells, where it is not possible tocontrol the timing and location of the release of siRNA and highconcentrations of transfection reagent are needed. By lowering thecarrier amount (changing the siRNA:carrier ratio) compared to the amountthat is advocated for use or by lowering the overall number ofsiRNA:carrier complexes that are applied to the cell it may also bepossible to minimise the siRNA leakage from the intracellularcompartments prior to PCI irradiation.

It has further been shown that by using the carriers as defined hereinwith PCI to deliver siRNA, strong gene silencing effects can be achievedwithout also causing cytotoxicity. For example, using PEI (Mw 25000) at1 μg/ml with 100 nM siRNA and light doses of up to 40 seconds, nocytotoxic effects were observed (see FIG. 11B). Under these conditions,strong gene silencing effects were observed (see FIG. 10).

RNA is a polymer of ribonucleotides, each containing the sugar ribose inassociation with a phosphate group and a nitrogenous base (typically,adenine, guanine, cytosine, or uracil). As is the case for DNA, RNA canform complementary hydrogen bonds, and RNA may be double-stranded(dsRNA), single-stranded (ssRNA) or double-stranded with asingle-stranded overhang. “Small interfering RNA” (siRNA) refers todouble-stranded RNA molecules from about 10 to about 30 nucleotides longthat specifically interfere with protein expression by binding to mRNAmolecules. Preferably, siRNA molecules are 12-28 nucleotides long, morepreferably 15-25 nucleotides long, still more preferably 19-23nucleotides long and most preferably 21-23 nucleotides long. Therefore,preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.

The length of one strand designates the length of an siRNA molecule. Forinstance, an siRNA that is described as 21 ribonucleotides long (a21-mer) could comprise two opposite strands of RNA that anneal togetherfor 19 contiguous base pairings. The two remaining ribonucleotides oneach strand would form an overhang. When an siRNA contains two strandsof different lengths, the longer of the strands designates the length ofthe siRNA. For instance, a dsRNA containing one strand that is 21nucleotides long and a second strand that is 20 nucleotides long,constitutes a 21-mer.

siRNAs that comprise an overhang are desirable. The overhang may be atthe 5′ or the 3′ end of a strand. Preferably, it is at the 3′ end of theRNA strand. The length of an overhang may vary, but preferably is about1 to about 5 nucleotides, and more preferably is about 2 nucleotideslong. Preferably, the siRNA of the present invention will comprise a 3′overhang of about 2 to 4 nucleotides. More preferably, the 3′ overhangis 2 ribonucleotides long. Even more preferably, the 2 ribonucleotidescomprising the 3′ overhang carry uracil (U) bases.

siRNAs are designed to interact with a target ribonucleotide sequence,in other words they complement a target sequence so as to bind to thetarget sequence, i.e. one strand of the siRNA is complementary to aregion of the target sequence.

siRNA molecules having modified backbones so as to increase their halflife have also been generated (e.g. as described in Chiu et al., (2003),RNA. 9(9), 1034-48 and Czauderna et al., (2003), Nucleic Acids Research31, 2705-2716). The term “siRNA” thus also includes such modifiedmolecules. Reference to siRNA thus encompasses derivatives or variantsof siRNA which exhibit the same function, i.e. interaction with a targetmRNA sequence. Preferred variants include those in which a modifiedbackbone has been used (as above) or one or more non-naturally occurringbases is used.

The method may be used to introduce more than one type of siRNA moleculeinto a cell. In other words, siRNA molecules having different sequencescan be introduced simultaneously into a cell. If multiple siRNAmolecules are to be introduced, this can be achieved by simultaneouslyassociating more than one siRNA molecule with the carrier.Alternatively, each type of siRNA molecule can separately be associatedwith a carrier.

There are several methods for preparing siRNAs, such as chemicalsynthesis, in vitro transcription, siRNA expression vectors, and PCRexpression cassettes. Such techniques are well known in the art. See forexample Pon et al., (2005) Nucleosides Nucleotides Nucleic Acids.24(5-7): 777-81, Du et al., (2006), Biochem. Biophys. Res. Commun.345(1):99-105 and Katoh et al., (2003), Nucleic Acids Res Suppl. (3):249-50.

Similarly, methods for designing siRNA molecules so as to achieve thedesired outcome have been well documented. The siRNA target site mustfirst be chosen. This can be carried out by using various techniques(see e.g. Jagla et al., (2005), RNA. 11(6):864-72 and Takasaki et al.,(2006), Comput. Biol. Chem. 30(3): 169-78).

The method of the invention achieves translocation of the siRNA moleculeinto the cytosol. It will be appreciated however that uptake of each andevery molecule contacted with the cell is not achievable. Significantand improved uptake relative to background levels in which no PCI orcarrier is used is however achievable.

Preferably methods of the invention allow the uptake of siRNA moleculesat sufficient levels that their effect is evident in the expressedproducts of those cells. The appropriate concentration of siRNA to becontacted with the cell may be adjusted to achieve this aim, e.g. toachieve a reduction in expression of a target gene of at least 10%, e.g.at least 20, 30, 40 50, 60, 70, 80 or 90% reduction (e.g. in theexpression of one or more proteins encoded by the target gene) afterincubation with cells for e.g. 24, 48, 72 or 96 hours (e.g. 24 to 48hours). Similarly, the carrier type and/or concentration, thephotosensitising agent type and/or concentration and the irradiationtime can be adjusted to achieve the reduction set out above.

This can be measured by determining the level of protein in the cell,using standard techniques known in the art such as Western Blotting. Thelevel of reduction of the protein is dependent on the half-life of theprotein, i.e. pre-existing protein will be removed in accordance withits half-life. Thus a reduction in expression of at least 10, 20, 30,40, 50, 60, 70, 80 or 90% is achieved relative to expression at the sametime point without siRNA so that half-life is taken into account.

This can alternatively be measured in terms of the effect of the siRNAmolecule on the amount of mRNA that is present in the cell, e.g. themethod can be carried out to achieve a reduction in mRNA levels of atleast 10%, e.g. at least 20, 30, 40, 50, 60, 70, 80 or 90% reductionafter incubation with cells for e.g. 24, 48, 72 or 96 hours e.g. 24 to48 hours) relative to mRNA levels of the target sequence at the sametime point without siRNA. This can also be measured using standardtechniques known in the art such as hybridisation or blotting techniquesand RT-PCR.

Since the present methods require the use of significantly less carrieror transfection agent (and/or less siRNA, depending on whether theoverall number of complexes is to be reduced, or the siRNA:carrier ortransfection agent ratio is to be modified, or both) than standardmethods for transfecting siRNA molecules, it is also possible to expressthe improvement in transfection using the method of the invention interms of the amount of carrier or transfection agent that is required toachieve a certain amount of reduction in protein expression or mRNAlevels. For example, the method of the invention preferably allows acertain reduction in target protein expression or mRNA levels (e.g. ofat least 10%, e.g. at least 20, 30, 40, 50, 60, 70, 80 or 90% asdescribed above) using a carrier concentration and/or siRNAconcentration that is e.g. at least 10, 20, 30, 40, 50 or 60% lower thanthe amount of carrier that is required to achieve the same level ofreduction in target protein expression or mRNA levels without PCI.

Comparisons can also be made between the levels of reduction in proteinexpression or mRNA levels that are seen at a certain siRNA and carrierconcentration, in the presence and absence of PCI. For example, themethod of the invention preferably allows a reduction in target proteinexpression or mRNA levels of at least 10%, e.g. at least 20, 30, 40, 50,60, 70, 80 or 90%, as described above, compared to protein expression ormRNA levels achieved by carrying out the method in the absence of theirradiation step of the PCI technique.

The term “cell” is used herein to include all eukaryotic cells(including insect cells and fungal cells). Representative “cells” thusinclude all types of mammalian and non-mammalian animal cells, plantcells, insect cells, fungal cells and protozoa. Preferably however thecells are mammalian, for example cells from cats, dogs, horses, donkeys,sheep, pigs, goats, cows, mice, rats, rabbits, guinea pigs, but mostpreferably from humans.

As used herein “contacting” refers to bringing the cells and thephotosensitizing agent and/or siRNA and carrier into physical contactwith one another under conditions appropriate for internalization intothe cells, e.g. preferably at 37° C. in an appropriate nutritionalmedium, e.g. from 25-39° C.

The photosensitising agent is an agent which is activated onillumination at an appropriate wavelength and intensity to generate anactivated species. Conveniently such an agent may be one which localisesto intracellular compartments, particularly endosomes or lysosomes. Arange of such photosensitising agents are known in the art and aredescribed in the literature, including in WO96/07432, which isincorporated herein by reference. Mention may be made in this respect ofdi- and tetrasulfonated aluminium phthalocyanine (e.g. AlPcS_(2a)),sulfonated tetraphenylporphines (TPPS_(n)), nile blue, chlorin e₆derivatives, uroporphyrin I, phylloerythrin, hematoporphyrin andmethylene blue which have been shown to locate in endosomes andlysosomes of cells in culture. This is in most cases due to endocyticuptake of the photosensitizer. Thus, the photosensitizing agent ispreferably an agent which is taken up into the internal compartments ofthe cell, e.g. lysosomes and/or endosomes. Further appropriatephotosensitizers for use in the invention are described in WO03/020309,which is also incorporated herein by reference, namely sulphonatedmeso-tetraphenyl chlorins, preferably TPCS_(2a).

However, other photosensitizing agents which locate to otherintracellular compartments for example the endoplasmic reticulum or theGolgi apparatus may also be used. It is also conceivable that mechanismsmay be at work in which the effects of the photochemical treatment areon other components of the cell (i.e. components other thanmembrane-restricted i.e. membrane enclosed compartments). Thus, forexample one possibility may be that the photochemical treatment destroysmolecules important for intracellular transport or vesicle fusion. Suchmolecules may not necessarily be located in membrane-restrictedcompartments, but the photochemical damage of such molecules maynevertheless lead to photochemical internalisation of the carrier:siRNAcomplexes, e.g. by a mechanism in which photochemical effects on suchmolecules leads to reduced transport of the molecule to be internalized(i.e. the siRNA molecule) to degradative vesicles such as lysosomes, sothat the molecule to be internalized can escape to the cytosol beforebeing degraded.

Examples of molecules not necessarily located in membrane restrictedcompartments are several molecules of the microtubular transport systemsuch as dynein and components of dynactin; and for example rab5, rab7,N-ethylmaleimde sensitive factor (NSF), soluble NSF attachment protein(SNAP) and so on.

Classes of suitable photosensitising agents which may be mentioned thusinclude porphyrins, phthalocyanines, purpurins, chlorins, (particularlychlorin derivatives of the prophyrins described below) benzoporphyrins,lysomotropic weak bases, naphthalocyanines, cationic dyes andtetracyclines or derivatives thereof (Berg et al., (1997), J.Photochemistry and Photobiology, 65, 403-409). Other suitablephotosensitising agents include texaphyrins, pheophorbides, porphycenes,bacteriochlorins, ketochlorins, hematoporphyrin derivatives, andderivatives thereof, endogenous photosensitizers induced by5-aminolevulinic acid and derivatives thereof, dimers or otherconjugates between photosensitizers.

Preferred photosensitising agents include TPPS₄, TPPS_(2a), AlPcS_(2a),TPCS_(2a) and other amphiphilic photosensitizers. Other suitablephotosensitizing agents include the compound 5-aminolevulinic acid oresters of 5-aminolevulinic acids or pharmaceutically acceptable saltsthereof.

“Irradiation” of the cell to activate the photosensitising agent refersto the administration of light directly or indirectly as describedhereinafter. Thus cells may be illuminated with a light source forexample directly (e.g. on single cells in vitro) or indirectly, e.g. invivo when the cells are below the surface of the skin or are in the formof a layer of cells not all of which are directly illuminated, i.e.without the screen of other cells.

In this method, the siRNA molecule to be introduced into the cell isattached to or associated with or conjugated to one or more carriermolecules or transfection agents which act to facilitate or increase theuptake of the photosensitizing agent or the siRNA molecule into thecell. This attachment, association or conjugation may be performed priorto contacting the siRNA molecule and its carrier with the cell or at thetime of said contact by virtue of bringing these molecules into contact.

The terms carrier and transfection agent are used interchangeablyherein.

The carrier molecule may be associated, bound or conjugated to the siRNAmolecule or to both the siRNA and the photosensitizing agent. Thus, forexample the siRNA may be attached to the carrier via charge:chargeinteractions. As mentioned above, more than one carrier may be usedsimultaneously, and the carrier may be associated, bound or conjugatedto more than one siRNA molecule, or more than one type of siRNAmolecule.

Preferably the carrier comprises a compound, preferably in anon-liposomal formulation that contains two or more amine groups i.e. isa polyamine and which is cationic and preferably protonatable (i.e. maybe protonated to carry one or more additional hydrogen atoms undersuitable reaction conditions) at different pH values. The different pHvalues applies to different values for protonatable atoms within asingle molecules and/or within different molecules.

The term “protonatable” is used herein to mean that a group is capableof accepting a hydrogen atom, i.e. a protonatable group is a hydrogenaccepting group. It is clear that the ability of a group to accepthydrogen depends not only on the nature of the group, but also on the pHto which the group is exposed. Preferably said protonatable groupcontains a nitrogen atom and it is this atom which accepts the hydrogenatom.

As referred to herein, “cationic” denotes that the overall, or net,charge of the molecule is +1 or higher. This is preferably measured atphysiological pH, i.e. pH 7.2. The molecule may have a higher charge,e.g. +2 or higher, +3 or higher, +4 or higher, +5 or higher, +6 orhigher, +7 or higher, +8 or higher, +9 or higher, +10 or higher, +11 orhigher, +12 or higher, +13 or higher, +14 or higher, +15 or higher, +20or higher, +25 or higher, +50 or higher, +75 or higher, +100 or higher,+150 or higher, +200 or higher, +250 or higher, +300 or higher, +400 orhigher, +500 or higher, +750 or higher or +1000 or higher.

Cationic polyamines for use in accordance with methods of the inventionare as defined hereinafter and include

-   -   (a) a lipopolyamine in a non-liposomal formulation,    -   (b) polyethyleneimine (PEI) having an M_(n) value of 500-20000        by GPC,    -   (c) a betacyclodextrin amine polymer of formula

wherein X is an integer from 1 to 100 inclusive and n is an integer from4 to 10 inclusive,

-   -   (d) an amine group containing dendrimer, and    -   (e) a cationic peptide.

Preferably, the polyamine as referred to herein contains primary orsecondary amine groups, or a mixture thereof (e.g. at least two primaryamine groups). Preferably, the polyamine region has at least 2, 3, 4, 5or 6 nitrogen atoms and a charge of at least +1, +2, +3, +4, or +5 (orat least +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +20, +25, +50,+75, +100, +150, +200, +250, +300, +400, +500, +750 or +1000) atphysiological pH, e.g. some or all of the amine groups are charged.Preferably at least one (e.g. at least 2, 3 or 4) nitrogen containinggroup e.g. NH is uncharged at physiological pH. The pKa at which thelast amine of the polyamine is protonated, e.g. the lipopolyamine ispreferably approximately 5.5, i.e. on decreasing the pH, or addingacidic compounds, the last amine to be protonated is protonated at a pHless than or equal to 5.5.

In one embodiment the carrier comprises a lipopolyamine in anon-liposomal formulation. By lipopolyamine it is meant an amphiphilicmolecule comprising at least one hydrophilic polyamine region (i.e.which contains two or more amine groups) and one lipophilic region. Thelipophilic region may contain one or more lipophilic chains.

The polyamine region of the lipopolyamine preferably has the formula (I)

in which m is an integer greater than or equal to 2 and n is an integergreater than or equal to 1, it being possible for m to vary between thedifferent carbon groups included between two amines, i.e. each(CH)_(m)—NH group may have a different m value, and m may be the same ordifferent where it appears in said formula. At each position R¹ is ahydrogen or a linking group to the lipo portion of the lipopolyamine orthe lipo portion itself as described hereinafter, and may be the same ofdifferent at each carbon atom. R² is a hydrogen or a linking group tothe lipo portion of the lipopolyamine or the lipo portion itself asdescribed hereinafter. Preferentially, m is between 2 and 6 inclusive,more preferably 3 or 4 and n is between 1 and 5 inclusive, morepreferably 3.

Preferably only one of R¹ and R² is a linker, a linker attached to thelipo portion of the polyamine, or the lipo portion of the polyamine.Preferably only one R¹ group is a linker, the linker attached to thelipo portion of the polyamine or the lipo portion of the polyamine. R²is preferably H.

When R¹ or R² is the lipo portion itself or is a linker which isattached to the lipo portion of the polyamine, formula (I) is thelipopolyamine. Thus formula (I) is the polyamine region only when R¹ orR² is not the lipo portion or a linking group to which a lipo portion isattached.

Still more preferentially, the polyamine region is represented by thefollowing formula

Wherein R² and R^(1a) to R^(1j) are as R¹ defined above, and preferablyR^(1a) is a linker and the remaining R¹ groups and R² are hydrogen.

The linking group is comprised of bonds that are stable under normalconditions.

Preferably R¹ or R² represents a hydrogen atom or a radical of generalformula II:

in which R³ and R⁴, which may be identical or different, each representa saturated aliphatic radical C_(p) H_(2p+2) or unsaturated aliphaticradical C_(p) H_(2p) or C_(p) H_(2p−2), p being an integer between 12and 22 inclusive, and R⁵ represents a hydrogen atom or an alkyl radicalcontaining 1 to 4 carbon atoms optionally substituted with a phenylradical.

Alternatively, R¹ or R² may each be a radical of general formula III:

in which X represents a methylene group (—CH₂—) or a carbonyl group(—CO—), and R⁶ and R⁷, which may be identical or different, eachrepresent a saturated aliphatic radical C_(p′) H_(2p′+2) or unsaturatedaliphatic radical C_(p′) H_(2p′) or C_(p′) H_(2p′−2), p′ being aninteger between 11 and 21 inclusive.

Irrespective of the values of m and n, only one of the symbols R¹ and R²can represent a radical of general formula (II) or (III),

When n is between 2 and 5, the values of m in the different fragments

may be identical or different.

In a preferred embodiment of formula (I) n is equal to 3 and the valuesof m in the fragments

are identical or different and represent 3 or 4, and either R¹ or R²represents: either a radical of general formula (II) in which R³ and R⁴each represent an alkyl radical containing 12 to 22 carbon atoms and R⁵represents a hydrogen atom, or either R¹ or R² represent a radical ofgeneral formula (III) in which R⁶—X— and R⁷—X— each represent analkanoyl radical containing 12 to 22 carbon atoms.

Especially preferred are 5-carboxyspermylglycinedioctadecylamide (DOGS)and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).

The synthesis of the above lipopolyamines is described in U.S. Pat. No.5,476,962.

Further examples of lipopolyamines for use in accordance with theinvention include, 2,3-dioleyl-oxy-N[2-sperminecarboxyyl-amido]ethyl-N,N-dimethyl-1-propanaminiumtrifluoracetate (DOS PA), 1,3-dioleoyloxy-2-(6-carboxyspermine) (DOSPER)and RPR-120535 (Ahmed et al (2005) Pharmaceutical Research 22(6),972-980). Structures of preferred lipopolyamines are set out in FIG. 7.

In DOSPA the linking group is

In DOSPER the linking group is

And the above structures thus represent further suitable examples oflinking groups.

The lipophilic region can be as defined for R³, R⁴, R⁵ or R⁶ above orany saturated or unsaturated hydrocarbon chain, cholesterol or othersteroid, a natural lipid or a synthetic lipid capable of forminglamellar or hexagonal phases. The length of the hydrocarbon chain may befrom 10 to 30 carbons in length, e.g. 12-28, 14-26, 16-24, 18-22 carbonsin length.

The carrier is preferably JetSI™ or JetSI-ENDO™, both of which areavailable from Polyplus transfection. Alternatively, the carrier may beTransfectam®, available from Promega.

By liposomal formulation it is meant that the cationic chargedamphiphile (i.e. the lipopolyamine) is combined with a neutral helperlipid such as DOPE (dioleoyl phosphatidylethanolamine), so as to formliposomes. As such, a non-liposomal formulation of a lipopolyamine is alipopolyamine containing formulation in which the lipopolyamine is notpresent in the form of a liposome. In other words, such formulations donot contain, in addition to the lipopolyamine, any neutral helperlipids. Examples of helper lipids are neutral phospholipids,cholesterol, glycerophosphoethanolamines and diacylglycerol. Preferablythe lipopolyamine formulation contains solely the lipopolyaminedescribed herein.

It is known that in general combining transfection agents with helperlipids such as DOPE will increase transfection efficiency, and hence itis surprising that an improved, and more selective degree of inhibitioncan be achieved by omitting these compounds from the formulation whenused in methods of the invention.

The carrier is preferably not lipofectamine 2000, lipofectin, jet PEI,or a carrier having the composition of these commercially availabletransfection reagents. The composition of lipofectamine 2000 is a 3:1(w/w) liposome formulation of the polycationic lipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA) (Chemical Abstracts Registry name:N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxpentyl-amino)ethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminiumtrifluoroacetate), and the neutral lipid dioleoylphosphatidylethanolamine (DOPE).

The composition of lipofectin is a 1:1 mix of DOTMA(1,2-dioleoyloxypropyl-3-trimethyl ammonium bromide) and DOPE(dioleoylphosphatidylethanolamine).

The carrier is also preferably not siPORT or a carrier having thecomposition of this commercially available transfection reagent.

In an alternative embodiment, the carrier is a polyamine compound whichis cationic (and preferably protonatable) at physiological pH, e.g.polyethyleneimine (PEI). PEI exists in many different structuralvariants, however variants having an M_(n) value (number averagemolecular weight) of 600 or more, by GPC (Gel Permeation Chromatography)are of most interest. For example, the PEI may have an M_(n) value of500-700, 500-750, 750-1000, 100-1250, 1000-1250, 1250-1500, 1000-20000,1100-15000, 1200-12500, 1250-10000, 1500-7500, 1750-5000, 2000-4000 or2500-3500.

The number average molecular weight is a way of determining themolecular weight of a polymer. Polymer molecules, even ones of the sametype, come in different sizes (chain lengths, for linear polymers), sothe average molecular weight will depend on the method of averaging. Thenumber average molecular weight is the common, mean, average of themolecular weights of the individual polymers. It is determined bymeasuring the molecular weight of n polymer molecules, summing theweights, and dividing by n.

${\overset{\_}{M}}_{n} = \frac{\sum\limits_{i}{N_{i}M_{i}}}{\sum\limits_{i}N_{i}}$

Linear forms of PEI or non-linear, e.g. branched PEI (which can forexample be of low molecular weight of the Mw values described herein),may be used.

By branched PEI it is meant PEI which contains tertiary amine groups, aswell as primary and secondary amine groups. The number of tertiary aminegroups, relative to the primary and/or secondary amine groups isindicative of the amount or degree of branching in the polymer. Ingeneral, branched PEI contains primary, secondary and tertiary aminegroups in a ratio of 0.5-1.5:1.5-2.5:0.5-1.5 e.g. 1:2:1 (i.e. a ratio of2:1 for the secondary and tertiary amine groups), but branched PEIs witha branching structure such that they contain relatively more or fewertertiary amine groups also exist and can be used in the presentinvention. Examples of alternative ratios are 1:1 to 3:1 (secondary totertiary amine groups) e.g. 1.2:1 to 2.8:1, 1.4:1 to 2.6:1, 1.6:1 to2.4:1, 1.8:1 to 2.2:1.

The molecular weight of the PEI is preferably lower than 30 kDa or 25kDa, e.g. lower than 15, 10, 5 or 2 kDa.

An example of suitable PEI is available from Sigma (408719Polyethylenimine (average Mw˜800 Da by LS, average Mn˜600 by GPC, Lowmolecular weight, water-free). Other commercially available PEI basedregents include Poly Sciences, Inc PEI (branched, Mw 10,000), USBiological Exgen 500, Polyplus transfection jetPEI™, Sigma ESCORT™VTransfection reagent, and Mirus TransIT-TKO®.

As described above, in a preferred embodiment, one or morebetacyclodextrin amine polymers can be used as the carrier molecule,i.e. the polyamine compound is a betacyclodextrin amine polymer.Suitable betacyclodextrin amine polymers and methods of synthesisingsuch molecules are described in Hwang et al., (2001) Bioconjugate Chem,12, 280-90.

Suitable betacyclodextrin amine polymers and a schematic showing theirsynthesis from appropriate monomers are set out below:

As set out above, n can be an integer from 4 to 10, inclusive,preferably 5 to 8 inclusive, or 6 to 7 inclusive. It is most preferably4, 6 or 8. X can be any integer. X is preferably 1 to 100, 10 to 50, 15to 25, 1 to 20, e.g. 2 to 15, 3 to 12, 4 to 10, 5 to 8 or 6 to 7inclusive. X is most preferably 4 or 5.

In a further preferred embodiment, one or more amine group containingdendrimers (e.g. a poly amido amide (PAMAM) dendrimer) can be used asthe carrier molecule, i.e. the polyamine compound is an amine groupcontaining dendrimer. Dendrimers represent a class of macromoleculararchitecture called “dense star” polymers.

Unlike classical polymers, dendrimers have a high degree of molecularuniformity, narrow molecular weight distribution, specific size andshape characteristics, and a highly-functionalized terminal surface.Dendrimers are thus artificially manufactured or synthesized molecules,which are built up from branched units or monomers to make amonodisperse, tree-like or generational structure. Synthesizingmonodisperse polymers demands a high level of synthetic control which isachieved through stepwise reactions, building the dendrimer up onemonomer layer, or “generation,” at a time. Each dendrimer consists of amultifunctional core molecule with a dendritic wedge attached to eachfunctional site. The core molecule is referred to as “generation 0.”Each successive repeat unit along all branches forms the nextgeneration, “generation 1,” “generation 2,” and so on until theterminating generation.

The manufacturing process is thus a series of repetitive steps startingwith a central initiator core. Each subsequent growth step represents anew “generation” of polymer with a larger molecular diameter, twice thenumber of reactive surface sites, and approximately doubles themolecular weight of the preceding generation. For example, thegeneration of the PAMAM dendrimer is described in Esfand et al., (2001)Drug Discovery Today, 6(8), 427-36 and in Kukowska-Latallo et al.,(1996), 93(10), 4897-902.

Suitable dendrimers include all dendrimers containing amine groups, e.g.dendrimers with triethanolamine, NH₃, or ethylenediame cores to whichamine containing monomers are attached. Particularly preferred are PAMAMdendrimers. Preerably the dendrimer is made up to polyamine monomers,e.g. having the general formula H₂N—(CH₂)_(m)—NH—(CO)_(n)—(CH₂)_(o)wherein m and o are integers from 1 to 10, preferably 1 or 2 and n is 0or 1

PAMAM dendrimers are shown in FIG. 14. Each “generation” represents theaddition of two new H₂N—CH₂—CH₂—NH—CO—CH₂—CH₂— groups to each of theterminal amino groups of the preceding generation; as also illustratedin the Figure.

The Table below shows the calculated properties of amine surfacefunctional PAMAM dendrimers by generation.

Molecular Measured Surface Generation Weight Diameter (A) Groups 0 51715 4 1 1,430 22 8 2 3,256 29 16 3 6,909 36 32 4 14,215 45 64 5 28,826 54128 6 58,048 67 256 7 116,493 81 512 8 233,383 97 1024 9 467,162 1142048 10 934,720 135 4096

Preferably the PAMAM dendrimer is of molecular weight 1000-235000 or3000-117000, e.g. 6000-60000 or 14000-30000 Da.

Dendrimers can also be defined with reference to their generation, andas such the dendrimer (e.g. the PAMAM dendrimer) is preferably ofgeneration 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, particularly generation2-6.

As mentioned above, in a further alternative embodiment, one or morecationic polypeptides can be used as the carrier molecule, i.e. thepolyamine compound is a cationic polypeptide. Various cationic peptidesare known in the art.

A “peptide” as defined herein includes any molecule containing anynumber of amino acids, i.e. one or more amino acids. Preferably howeverthe peptide is a polymer of consecutive amino acids.

The peptides may be prepared by any convenient means, e.g. directchemical synthesis or by recombinant means by expressing a nucleic acidmolecule of the appropriate sequence in a cell.

As referred to herein in reference to peptides, “cationic” denotes thatthe overall, or net, charge of the peptide is +1 or higher atphysiological pH, i.e. pH 7.2. An amino acid is considered +1 if thepredominant species at physiological pH is positively charged whenpresent in the context of the peptide. Each such amino acid in a peptidecontributes a further positive charge to calculate the final charge ofthe peptide. The peptide may contain one or more negatively chargedamino acid residues, as well as neutral residues, as long as the netcharge of the peptide (calculated by adding together the chargeattributed to each amino acid) is positive.

The charge of the peptide therefore depends on its amino acidcomposition. Certain amino acids are charged at normal physiological pH.Positively charged amino acids are lysine (K), arginine (R) andhistidine (H) and are considered to be +1 on the above-described scale.Aspartic acid (D) and glutamic acid (E) carry a negative charge at mostphysiological pHs and are considered −1 on the above scale. Othernaturally occurring amino acids are considered to carry no charge. Anynumber of positively charged or negatively charged amino acids may bepresent, as long as the overall charge of the peptide is +1 or more.

The amino acids used in peptides for use in the invention need notnecessarily be naturally occurring amino acids. One of more of the aminoacids in the peptide may be substituted for a non-naturally occurring,e.g. a derivatized amino acid. Such amino acids would similarly beassessed on the basis of their contribution to the charge of thepeptide. Thus, as with naturally occurring amino acids, if thepredominant species is positive at physiological pH, whether or not thatcharge is derived from the derivatized portion (e.g. an introduced aminegroup) or a portion also present in the natural amino acid is irrelevantas long as the overall charge is +1 or more.

The charge of the peptide is >1, preferably from +1 to +1000, from +1 to+500, from +1 to 250 or from +1 to +100, e.g. +2 to +80, such as +3 to+60 or +4 to +50, +5 to +30, +6 to +20 e.g. +10 or +15.

Preferably the cationic peptide comprises L or D lysine, L or Darginine, L or D histidine and/or ornithine residues. Even morepreferably the peptide is rich in one or more of these residues e.g.comprises 10-100%, 20-80%, 30-70%, 40-60% or 50% positively chargedresidues. Examples of such peptides include Poly-L-Lysine,Poly-D-Lysine, Poly-Histidine, Histidylated poly-lysine andPoly-ornithine or copolymers of L or D lysine, L or D arginine, L or Dhistidine and/or ornithine residues with other amino acids e.g. one ormore of Alanine, Asparagine, Aspartic acid, Cysteine, Glutamic acid,Glutamine, Glycine, Isoleucine, Leucine, Methionine, Phenylalanine,Proline, Serine, Threonine, Tryptophan, Tyrosine and Valine.

Said cationic polypeptides can be defined in terms of their molecularweight. As such they are preferably at least 1000 Da, 1500 Da, 2000 Da,2500 Da, 5000 Da, 7500 Da, 10000 Da, 15000 Da, 20000 Da, 25000 Da, 30000Da, 40000 Da, 50000 Da, 60000 Da, 70000 Da, 80000 Da, 90000 Da or 100000Da in weight.

Alternatively the cationic polypeptides can be defined in terms of theirlength. Preferred cationic polypeptides are at least 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500 aminoacids in length.

A highly preferred cationic peptides is polyarginine, particularlypolyarginine with a Mw of at least 15000 kDa.

Although not wishing to be bound by theory, the association of thecarrier with the siRNA molecule is thought to be based on theinteraction of the positively charged carrier with the negativelycharged siRNA molecule, leading to the formation of an siRNA-carriercomplex. The siRNA-carrier complexes interact with anionic proteoglycansat the cell surface and are taken up by endocytosis.

In general the association, binding or conjugation of the carrier withthe siRNA molecule or both the siRNA and the photosensitizing agent isachieved by simply mixing the two components under appropriateconditions and concentrations and allowing the components to interact.In a preferred embodiment therefore the method comprises the additionalstep of contacting said siRNA with said carrier. The conditions underwhich this contacting step is carried out, and appropriateconcentrations for each of the carrier and the siRNA molecule canreadily be determined by the person skilled in the art by carrying outroutine testing. Examples of suitable conditions are as set out in theExamples. For example, the siRNA molecule and the carrier molecule maybe mixed together, e.g. by vortexing, and allowed to stand, e.g. at roomtemperature. The siRNA molecule and the carrier molecule may then beleft for approximately 10-20, 10-30 or 20-40 minutes before beingcontacted with the cell.

Preferably 10 nM-200 nM, e.g. 15-150 nM, 20-100 nM, 20-100 nM, 30-90 nM,40-80 nM, or 50-70 nM siRNA is used for transfection, e.g. in a well ofa standard 6 well plate, although different concentrations may betested. It is a matter of routine to determine the optimum concentrationof siRNA to be used in the method.

The cells to which the siRNA and carrier are applied are prepared usingstandard cell culture techniques. If the cells are adherent cells theyare preferably at 50-70% or 25-50% confluence.

The carrier and the siRNA molecule may be mixed in various ratios, basedon standard protocols. For example, as set out in Examples 1 to 6, 8.4μl of 2 mM carrier made up in an aqueous solution can be mixed with 2.8μg siRNA in 2 ml medium for application to a 6 well plate. Similarly,4.2 μl of 2 mM carrier made up in an aqueous solution can be mixed with1.4 μg siRNA in 1 ml medium for application to a 6 well plate. Thecarrier need not always be at 2 mM, suitable ranges include 0.5-5 mM,1-3 mM, 1.5-2.5 mM (e.g. made up in an aqueous solution). For every ngof siRNA, an amount equivalent to approximately 6 nanomoles of carrier(e.g. 1-10, 2-8) total in the solution with the photosensitiser can beused.

For PEI, preferred concentrations are 1 μg/ml and 10 μg/ml (e.g. 0.5-20or 1-15 μg/ml) with 100 nM siRNA, e.g. in 1 ml medium for application toa 6 well plate. This has been shown to be particularly effective for PEIwith a molecular weight of 1200-25000 Daltons (e.g. 1300-2000 Daltons)as set out in Example 9. The concentration of PEI used can be modifiedto achieve the charge ratio and/or N/P ratio that is achieved using theconditions set out in Example 9.

For betacyclodextrin amine polymers, a suitable concentration is 100μg/ml (e.g. 10-1000, 20-800, 30-600, 40-400, 50-200 μg/ml) with 50 nMsiRNA, e.g. in 1 ml medium for application to a 6 well plate. This isbased on the use of the polymer as described in Example 11. The valuesof n and X will influence the charge of the molecule and as suchdifferent concentrations may be appropriate. The concentration ofbetacyclodextrin amine polymer used can be modified to achieve thecharge ratio and/or N/P ratio that is achieved using the conditions setout in Example 11.

For amine group containing dendrimers, a suitable concentration is 100μg/ml (e.g. 10-1000, 20-800, 30-600, 40-400, 50-200 μg/ml) with 100 nMsiRNA, e.g. in 1 ml medium for application to a 6 well plate. Theconcentration of amine group containing dendrimer used can be modifiedto achieve the charge ratio and/or N/P ratio that is achieved using theconditions set out in Example 12.

For cationic peptides, a suitable concentration is 0.35 μg/ml or 0.7μg/ml (e.g. 0.1-20, 0.2-15, 0.3-10 μg/ml) with 100 nM siRNA, e.g. in 1ml medium for application to a 6 well plate. As set out in Example 13,these concentrations were used successfully with polyarginine carrier ofmolecular weight 15000-70000 and >70000. The molecular weight and aminoacid composition of the peptide will influence the charge of themolecule and as such different concentrations may be appropriate. Theconcentration of cationic peptide used can be modified to achieve acharge ratio and/or N/P ratio as is achieved using the conditions setout in Example 13.

The ratio of the two components may be expressed as the ratio ofcharges, and this also needs to be taken into account. Preferably thecharge ratio between the carrier and the siRNA is at least 1+/− (i.e. 1positive charge per negative charge), 5+/−, 10+/, 20+/−, 30+/−, 40+/−,50+/−, 60+/−, 70+/−, 80+/−, 90+/−, 100+/−, 200+/−, 300+/−, 400+/− or500+/−.

The charge ratio depends on the charge of each component (i.e. the siRNAand the carrier) and on the amount of each component that is present.

Alternatively, the ratio of the two components may be expressed as theN/P ratio, i.e. the ratio of nitrogen residues to oligonucleotidephosphate. Since not every nitrogen atom of a carrier is always acation, the N/P ratio is not the same as the charge ratio. The N/P ratiodepends on the chemical composition of each compound and on the amountof each compound that is present. Suitable values for the N/P ratioinclude 1-500, e.g. 2-450, 3-400, 4-350, 5-300, 6-250, 7-200, 8-150,9-100, 10-80, 15-60, 20-50, 30-40, 1-25, 1-20, 1-15, 1-10, 1-5.

Preferably the carrier is such that at the concentration that is chosenfor use, there is no release from intracellular compartments without theirradiation step of PCI, and release from intracellular compartments isseen following the irradiation step of PCI. Suitable concentrations andratios of siRNA and carrier are mentioned above.

The carrier is in general formulated for transfection purposes in anaqueous solution (e.g. in water). Stock solution (e.g. 20 mM in Ethanol)can then be diluted (e.g. in water) to an appropriate concentration foruse. U.S. Pat. No. 5,476,962 describes the formulation oflipopolyamines.

As mentioned above, the siRNA carrier may also be used as a carrier forthe photosensitising agent. Alternatively, however the photosensitisermay be used without a carrier or with an alternative carrier not usedaccording to the invention for siRNA. Such alternative carriers arereferred to herein as photosensitizer carriers and include polycationssuch as polylysine (e.g. poly-L-lysine or poly-D-lysine),polyethyleneimine or dendrimers (e.g. cationic dendrimers such asSuperFect7); cationic lipids such as DOTAP or Lipofectin and peptides.

In order to target the siRNA molecule and/or the photosensitizing agentto specific cells (e.g. cancer cells) or tissues, the siRNA moleculeand/or the photosensitizer and/or the carrier may be associated orconjugated to specific targeting molecules that will promote thespecific cellular uptake of the siRNA molecule into desired cells ortissues.

Many different targeting molecules can be employed, e.g. as described inCuriel (1999), Ann. New York Acad. Sci. 886, 158-171; Bilbao et al.,(1998), in Gene Therapy of Cancer (Walden et al., eds., Plenum Press,New York); Peng and Russell (1999), Curr. Opin. Biotechnol. 10, 454-457;Wickham (2000), Gene Ther. 7, 110-114.

The targeting molecule may be associated, bound or conjugated to thesiRNA molecule, to the carrier, to the photosensitising agent or to two(e.g. the siRNA and the carrier or the siRNA and the photosensitisingagent or the carrier and the photosensiting agent) or all three of thesemoieties, and the same or different targeting molecules may be used. Asmentioned above, more than one targeting molecule may be usedsimultaneously.

The method of the invention may be put into practice as described below.In the method of the invention, the siRNA molecule, together with itscarrier and a photosensitising compound (optionally with the samecarrier or with a photosensitizer carrier) are applied simultaneously orin sequence to the cells, whereupon the photosensitizing compound,carrier and the siRNA molecule are endocytosed or in other waystranslocated into endosomes, lysosomes or other intracellular membranerestricted compartments.

The siRNA, carrier and the photosensitising compound may be applied tothe cells together or sequentially. In general the siRNA is mixed withthe carrier as described above so as to form a complex, which is thenadministered to the cell simultaneously with the photosensitisingcompound. Alternatively, the siRNA:carrier complex and thephotosensitising compound can be administered sequentially. ThesiRNA-carrier complex and the photosensitising compound may be taken upby the cell into the same or different intracellular compartments (e.g.they may be co-translocated).

The siRNA is then released by exposure of the cells to light of suitablewavelengths to activate the photosensitising compound which in turnleads to the disruption of the intracellular compartment membranes andthe subsequent release of the siRNA, which may be located in the samecompartment as the photosensitizing agent, into the cytosol. Thus, inthese methods the final step of exposing the cells to light results inthe siRNA being released from the same intracellular compartment as thephotosensitizing agent and becoming present in the cytosol.

WO 02/44396 (which is incorporated herein by reference) describes amethod in which the order of the steps could be changed such that forexample the photosensitising agent is contacted with the cells andactivated by irradiation before the molecule to be internalised (and thecarrier) are brought into contact with the cells. This adapted methodtakes advantage of the fact that it is not necessary for the molecule tobe internalised to be present in the same cellular subcompartment as thephotosensitising agent at the time of irradiation.

Thus in a preferred embodiment, said photosensitising agent, saidcarrier and said siRNA molecule (e.g. as a carrier:siRNA complex) areapplied to the cell together, or said photosensitising agent is appliedseparately relative to said carrier and said siRNA molecule. As aconsequence they may be taken up by the cell into the same intracellularcompartment and said irradiation may then be performed. Thephotosensitising agent, carrier and siRNA molecule can be separate, orthey can be formulated as a dendrimeric molecule (see e.g. Nishiyama Net al., (2005) Nat. Mater. 4(12):934-41)

In an alternative embodiment, said method can be performed by contactingsaid cell with a photosensitising agent, contacting said cell with thecarrier and the siRNA molecule to be introduced and irradiating saidcell with light of a wavelength effective to activate thephotosensitising agent, wherein said irradiation is performed prior tothe cellular uptake of said siRNA molecule and said carrier into anintracellular compartment containing said photosensitising agent,preferably prior to cellular uptake of said molecule and said carrierinto any intracellular compartment.

Said irradiation can be performed after the cellular uptake of themolecule and the carrier molecule into an intracellular compartment,whether or not said siRNA molecule and the photosensitising agent arelocalised in the same intracellular compartments at the time of lightexposure. In one preferred embodiment however irradiation is performedprior to cellular uptake of the molecule to be internalised.

“Internalisation” as used herein, refers to the cytosolic delivery ofmolecules. In the present case “internalisation” thus includes the stepof release of molecules from intracellular/membrane bound compartmentsinto the cytosol of the cells.

As used herein, “cellular uptake” or “translocation” refers to one ofthe steps of internalisation in which molecules external to the cellmembrane are taken into the cell such that they are found interior tothe outer lying cell membrane, e.g. by endocytosis or other appropriateuptake mechanisms, for example into or associated with intracellularmembrane-restricted compartments, for example the endoplasmic reticulum,Golgi body, lysosomes, endosomes etc.

The step of contacting the cells with a photosensitising agent and withthe siRNA molecule and carrier may be carried out in any convenient ordesired way. Thus, if the contacting step is to be carried out in vitrothe cells may conveniently be maintained in an aqueous medium such asfor example appropriate cell culture medium and at the appropriate timepoint the photosensitising agent and/or siRNA molecule and carrier cansimply be added to the medium under appropriate conditions, for exampleat, an appropriate concentration and for an appropriate length of time.For example, the cells may be contacted with the siRNA molecule andcarrier in the presence of serum-free medium.

The photosensitizing agent is brought into contact with the cells at anappropriate concentration and for an appropriate length of time whichcan easily be determined by a skilled person using routine techniquesand will depend on such factors as the particular photosensitizing agentused and the target cell type and location. The concentration of thephotosensitizing agent must be such that once taken up into the cell,e.g. into, or associated with, one or more of its intracellularcompartments and activated by irradiation, one or more cell structuresare disrupted e.g. one or more intracellular compartments are lysed ordisrupted. For example photosensitising agents as described herein maybe used at a concentration of for example 10 to 50 μg/ml. For in vitrouse the range can be much broader, e.g. 0.05-500 μg/ml. For in vivohuman treatments the photosensitizing agent may be used in the range0.05-20 mg/kg body weight when administered systemically or 0.1-20% in asolvent for topical application. In smaller animals the concentrationrange may be different and can be adjusted accordingly.

The time of incubation of the cells with the photosensitizing agent(i.e. the “contact” time) can vary from a few minutes to several hours,e.g. even up to 48 hours or longer, e.g. 12 to 20 or 24 hours. The timeof incubation should be such that the photosensitizing agent is taken upby the appropriate cells, e.g. into intracellular compartments in saidcells.

The incubation of the cells with the photosensitizing agent mayoptionally be followed by a period of incubation withphotosensitiser-free medium before the cells are exposed to light or thesiRNA molecule and carrier are added, e.g. for 10 minutes to 8 hours,especially 1 to 4 hours.

The siRNA molecule and carrier (e.g. as a preformed siRNA:carriercomplex) are brought into contact with the cells at an appropriateconcentration and for an appropriate length of time.

Determining the appropriate doses of siRNA molecules for use in themethods of the present invention is routine practice for a personskilled in the art. For in vitro applications an exemplary dose of thesiRNA molecules would be approximately 1-100 nM siRNA and for in vivoapplications approximately 10⁻⁶−1 g siRNA per injection in humans. Forexample, siRNA molecules may be administered at levels of less than 500nM, e.g. less than 300 nM, especially preferably less than 100 nM or 50nM, for example from 1 to 100 nM, or 5 to 50 nM, where the concentrationindicated reflects the levels in contact with the cell.

As mentioned above, it has been found that the contact may be initiatedeven several hours after the photosensitising agent has been added andirradiation taken place.

An appropriate concentration can be determined depending on theefficiency of uptake of the siRNA molecule in question into the cells inquestion and the final concentration it is desired to achieve in thecells. Thus “transfection time” or “cellular uptake time” i.e. the timefor which the molecules are in contact with the cells can be a fewminutes or up to a few hours, for example a transfection time of from 10minutes until up to 24 hours, for example 30 minutes up to 10 hours orfor example 30 minutes until up to 2 hours or 6 hours can be used.Longer incubation times may also be used, e.g. 24 to 96 hours or longer,e.g. 5-10 days.

An increased transfection time usually results in increased uptake ofthe molecule in question. However, shorter incubation times, for example30 minutes to 1 hour, can also result in an improved specificity of theuptake of the molecule. Thus, in selecting a transfection time for anymethod, an appropriate balance must be struck between obtaining asufficient uptake of the molecule while maintaining sufficientspecificity of the PCI treatment.

In vivo an appropriate method and time of incubation by which the siRNAmolecule, carrier and photosensitizing agents are brought into contactwith the target cells will be dependent on factors such as the mode ofadministration and the type of siRNA molecule, carrier andphotosensitizing agents. For example, if the siRNA molecule and carrierare injected into a tumour, tissue or organ which is to be treated, thecells near the injection point will come into contact with and hencetend to take up the siRNA molecule more rapidly than the cells locatedat a greater distance from the injection point, which are likely to comeinto contact with the siRNA molecule at a later time point and lowerconcentration.

In addition, an siRNA molecule administered by intravenous injection maytake some time to arrive at the target cells and it may thus take longerpost-administration e.g. several days, in order for a sufficient oroptimal amount of the siRNA molecule to accumulate in a target cell ortissue. The same considerations of course apply to the time ofadministration required for the uptake of the photosensitizing agentinto cells. The time of administration required for individual cells invivo is thus likely to vary depending on these and other parameters.

Nevertheless, although the situation in vivo is more complicated than invitro, the underlying concept of the present invention is still thesame, i.e. the time at which the molecules come into contact with thetarget cells must be such that before irradiation occurs an appropriateamount of the photosensitizing agent has been taken up by the targetcells and either: (i) before or during irradiation the siRNA moleculehas either been taken up, or will be taken up after sufficient contactwith the target cells, into the same or different intracellularcompartments or (ii) after irradiation the siRNA molecule is in contactwith the cells for a period of time sufficient to allow its uptake intothe cells. Provided the siRNA molecule is taken up into intracellularcompartments affected by activation of the photosensitizing agent (e.g.compartments in which the agent is present), the siRNA molecule can betaken up before or after irradiation.

The light irradiation step to activate the photosensitising agent maytake place according to techniques and procedures well known in the art.For example, the wavelength and intensity of the light may be selectedaccording to the photosensitising agent used. Suitable light sources arewell known in the art.

The time for which the cells are exposed to light in the methods of thepresent invention may vary. The efficiency of the internalisation of thesiRNA molecule into the cytosol increases with increased exposure tolight to a maximum beyond which cell damage and hence cell deathincreases.

A preferred length of time for the irradiation step depends on factorssuch as the target, the photosensitizer, the amount of thephotosensitizer accumulated in the target cells or tissue and theoverlap between the absorption spectrum of the photosensitizer and theemission spectrum of the light source. Generally, the length of time forthe irradiation step is in the order of minutes to several hours, e.g.preferably up to 60 minutes e.g. from 0.5 or 1 to 30 minutes, e.g. from0.5 to 3 minutes or from 1 to 5 minutes or from 1 to 10 minutes e.g.from 3 to 7 minutes, and preferably approximately 3 minutes, e.g. 2.5 to3.5 minutes. Shorter irradiation times may also be used, for example 1to 60 seconds, e.g. 10-50, 20-40 or 25-35 seconds.

Appropriate light doses can be selected by a person skilled in the artand again will depend on the photosensitizer and the amount ofphotosensitizer accumulated in the target cells or tissues. For example,the light dose typically used for photodynamic treatment of cancers withthe photosensitizer Photofrin and the protoporphyrin precursor5-aminolevulinic acid is in the range 0.50-150 J/cm2 at a fluence rangeof less than 200 mW/cm² in order to avoid hyperthermia. The light dosesare usually lower when photosensitizers with higher extinctioncoefficients in the red area of the visible spectrum are used. However,for treatment of non-cancerous tissues with less photosensitizeraccumulated the total amount of light needed may be substantially higherthan for treatment of cancers. Furthermore, if cell viability is to bemaintained, the generation of excessive levels of toxic species is to beavoided and the relevant parameters may be adjusted accordingly.

The methods of the invention may inevitably give rise to some cellkilling by virtue of the photochemical treatment i.e. through thegeneration of toxic species on activation of the photosensitizing agent.Depending on the proposed use, this cell death may not be of consequenceand may indeed be advantageous for some applications (e.g. cancertreatment). Preferably, however, cell death is avoided and as notedelsewhere herein the method can be carried out so as to cause stronginhibition of expression (i.e. a strong siRNA effect) in the absence ofcell toxicity. It is highly advantageous to achieve a strong inhibitionof expression in the absence of general cell toxicity or an effect oncell viability. The methods of the invention may be modified such thatthe fraction or proportion of the surviving cells is regulated byselecting the light dose in relation to the concentration of thephotosensitizing agent. Again, such techniques are known in the art.

In applications in which viable cells are desirable, substantially allof the cells, or a significant majority (e.g. at least 50%, morepreferably at least 60, 70, 80 or 90% of the cells) are not killed. Cellviability following PCI treatment can be measured by standard techniquesknown in the art such as the MTS test (see the Examples).

Regardless of the amount of cell death induced by the activation of thephotosensitiser, for the siRNA to have an effect in the cells, it isimportant that the light dose is regulated such that some of theindividual cells wherein the PCI effect is manifested are not killed bythe photochemical treatment alone (although they may subsequently bekilled by molecules introduced into the cells if those molecules, have acytotoxic effect).

Cytotoxic effects may be achieved by using for example gene therapy inwhich an siRNA molecule is internalized into a tumour cell by the methodof the invention e.g. to down-regulate a gene.

The methods of the invention may be used in vitro or in vivo, forexample either for in situ treatment or for ex vivo treatment followedby the administration of the treated cells to the body, for variouspurposes including inhibition of expression of specific gene productse.g. in gene therapy methods and the generation of screening assays.

Thus the present invention provides a method of inhibiting theexpression of a target gene by introducing an siRNA molecule into a cellcontaining said target gene by a method as described hereinbefore,wherein said siRNA molecule specifically inhibits expression of saidtarget gene.

“Specific inhibition” refers to sequence-dependent inhibition of thetarget gene. Expression of genes which contain a sequence that issufficiently identical at the nucleic acid level to the siRNA moleculeused will be affected by the siRNA molecule. As noted above, standardtechniques have been developed that allow the skilled person to designsiRNA molecules of appropriate sequence to cause sequence-specificinhibition of expression.

“Target gene” refers to a gene whose expression is to be down-regulatedand which is to be the target of investigation or manipulation.

These methods may be used to alter the expression profile of cells, e.g.to investigate cellular pathways, or to determine the influence ofexpression of a particular gene, or for therapeutic purposes.

The methods of the invention may also be used in treating any diseasewhich benefits from the down-regulation, repair or mutation of one ormore genes. For example, genes that are overexpressed in cancer may bedown-regulated by administering the appropriate siRNA molecule (Lage(2005) Future Oncol 1(1):103-13). Alternative diseases that may betreated include neurodegenerative diseases such as Huntington's diseaseand Alzheimer's disease and viral infections such as hepatitis (e.g. Band C) and HIV.

Thus, a further aspect of the invention provides a compositioncontaining an siRNA molecule, a carrier molecule (preferably as acomplex with said siRNA molecule) and optionally separately also aphotosensitizing agent as described herein. In a further aspect theinvention provides said composition for use in therapy.

Alternatively, the present invention provides a kit comprising an siRNAmolecule, a carrier molecule and optionally also a photosensitizingagent as described herein. Preferably said kit (or product) is forsimultaneous, separate or sequential use in a medical treatment.

Alternatively described, the present invention provides the use of ansiRNA molecule and carrier as described herein in the preparation of amedicament for treating or preventing a disease, disorder or infectionby altering expression of one or more target genes in said patient.Optionally said medicament may contain only one of said siRNA moleculeor carrier and may be used in methods in which said siRNA molecule orcarrier not present in said medicament is for administration to saidpatient when treating or preventing said disease, disorder or infection.Optionally said medicament may contain the photosensitising agent.Preferably said medicament is for gene therapy, i.e. for treating adisease or disorder which is typified by abnormal gene expression orwhich would benefit from suppression of one or more genes. Saidalteration includes down regulation of said expression.

According to the different embodiments set out above, the saidphotosensitizing agent and said siRNA molecule and carrier is contactedwith cells or tissues of a patient simultaneously or sequentially andsaid cells are irradiated with light of a wavelength effective toactivate the photosensitizing agent and irradiation is performed priorto, during or after the cellular uptake of said siRNA molecule and thecarrier into an intracellular compartment containing saidphotosensitizing agent, preferably prior to cellular uptake of saidtransfer molecule into any intracellular compartment.

Thus in an alternative aspect the invention provides a method oftreating or preventing a disease, disorder or infection in a patientcomprising introducing an siRNA molecule and carrier into one or morecells in vitro, in vivo or ex vivo according to the methods as describedhereinbefore and where necessary (i.e. when transfection is conducted invitro or ex vivo) administering said cells to said patient.

As defined herein “treatment” refers to reducing, alleviating oreliminating one or more symptoms of the disease, disorder or infectionwhich is being treated, relative to the symptoms prior to treatment.“Prevention” refers to delaying or preventing the onset of the symptomsof the disease, disorder or infection.

Compositions of the present invention may also comprise a cellcontaining an siRNA molecule which has been internalised into thecytosol of said cell by a method of the invention. The invention furtherextends to such compositions for use in therapy, particularly cancer orgene therapy.

Thus, a yet further aspect of the invention provides a cell or apopulation of cells containing an siRNA molecule which has beeninternalised into the cytosol of said cell, which cell is obtainable bya method of the present invention.

A yet further aspect of the invention provides the use of a such a cellor population of cells for the preparation of a composition or amedicament for use in therapy as described hereinbefore, preferablycancer or gene therapy.

The invention further provides a method of treatment of a patientcomprising administering to said patient cells or compositions of thepresent invention, i.e. a method comprising the steps of introducing ansiRNA molecule into a cell as described hereinbefore and administeringsaid cell thus prepared to said patient. Preferably said methods areused to treat cancer or in gene therapy.

In vivo, any mode of administration common or standard in the art may beused, e.g. injection, infusion, topical administration, both to internaland external body surfaces etc. For in vivo use, the invention can beused in relation to any tissue which contains cells to which thephotosensitising agent and the siRNA molecule are localized, includingbody fluid locations, as well as solid tissues. All tissues can betreated as long as the photosensitiser is taken up by the target cells,and the light can be properly delivered.

Thus, the compositions of the invention may be formulated in anyconvenient manner according to techniques and procedures known in thepharmaceutical art, e.g. using one or more pharmaceutically acceptablecarrier or excipients. “Pharmaceutically acceptable” as referred toherein refers to ingredients that are compatible with other ingredientsof the compositions as well as physiologically acceptable to therecipient. The nature of the composition and carriers or excipientmaterials, dosages etc. may be selected in routine manner according tochoice and the desired route of administration, purpose of treatmentetc. Dosages may likewise be determined in routine manner and may dependupon the nature of the molecule, purpose of treatment, age of patient,mode of administration etc. In connection with the photosensitizingagent the potency/ability to disrupt membranes on irradiation, shouldalso be taken into account.

The methods described above may alternatively be used to generate ascreening tool for high throughput screening methods, particularly toanalyze the effects of silencing a particular gene. siRNA directed toone or more specific genes may be generated and used in the method ofthe invention as described above. The siRNA may thus be used to reducethe expression of a gene in a population of cells. The resulting cellpopulation may then be used as a screening tool to identify downstreameffects of gene silencing, with standard techniques.

Previous attempts to reduce gene expression with normal and chemicallymodified antisense oligonucleotides have been limited by problems withnuclease degradation of the antisense oligonucleotides, the occurrenceof non-specific effects and/or insufficient target affinity. By usingthe method of the invention to administer siRNA, these problems may beovercome.

Thus in a further aspect, the invention provides a method of modifyingthe gene expression pattern of a cell (e.g. a cell population) toprepare a cell (or cell population) for use as a screening tool (e.g.for high throughput screening), comprising contacting an siRNA moleculecapable of inhibiting or reducing the expression of a gene, a carrierand a photosensitising agent with a cell (e.g. a cell population) andirradiating the cell (e.g. a cell population) with light of a wavelengtheffective to activate the photosensitising agent. The invention furtherextends to such cells and a method of screening such cells whereinspecific properties of such cells, e.g. mRNA expression levels of suchcells are examined, e.g. in microarrays. By “modified gene expressionpattern” it is meant that as a consequence of the presence of said siRNAmolecule in the cell nucleus, the transcription or translation of thegene to which it is directed is affected.

As a consequence of this change in expression of the gene, theexpression of other genes may be influenced. Thus, by affecting thenormal expression of the gene being studied, it is possible to determinethe changes in expression pattern of other genes. The identification ofthese genes, and of the influence that the expression of the gene beingstudied has on them allows the investigator to draw conclusions aboutthe functions of the gene e.g. their downstream functions. The genesthat are affected by the change in normal expression of the gene beingstudied may be upregulated or downregulated, but the overall change inthe pattern of expression gives an indication of the role of the gene innormal cell function and of the consequences of its misregulation.

Using standard techniques well known in the art it is possible to studythe effect of the downregulation or elimination of expression of thegene in question. This may for example be done by looking for functionalchanges in the cells (or cell population), such as changes in celladhesion, protein secretion or morphological changes. Alternatively, thegene expression profile can be studied directly by analysing mRNApatterns and/or protein expression, again using standard techniques thatare well known in the art.

By inhibiting or reducing the expression of a gene, it is to beunderstood that the expression of the gene in question is reduced, whencompared to a cell which has not been subjected to the method i.e. awild-type or normal cell. The change in the level of gene expression maybe determined by standard techniques known in the art.

There may be a complete inhibition of expression, such that there is nodetectable expression of the gene, i.e. no mRNA or protein isdetectable, or there may be a partial inhibition of expression, i.e. areduction, whereby the amount of gene expression is lower than thewild-type or normal cell. This can be assessed and controlled for bycomparing the effect of an siRNA with a specific sequence with theeffect of an siRNA with a scrambled sequence i.e. the same compositionof nucleotides, but in a different sequence order. Preferably for thistechnique to be useful, the reduction in expression is to less than or80% of control levels, e.g. <50%, preferably <20, 10 or 5% of controllevels. The cell(s) used will preferably be a cell population, theindividual cells of which are genetically identical. The cells may beany cells, as discussed above.

The cell or cell population generated according to methods of theinvention may be used to make a library which forms a further aspect ofthe invention.

The invention will now be described in more detail in the followingnon-limiting Examples with reference to the following drawings in which:

FIG. 1 shows the results of a gene silencing experiment using the OHScell line with various transfection reagents with (black bars) andwithout (white bars) PCI treatment using siRNA9. Graph displays S100A4protein levels, from left to right; 1) siPORT Lipid, 2) FuGene 6, 3)Lipofectamine 2000, 4) Lipofectin, 5) jetSI and 6) jetSI-ENDO. Resultsare represented as percent of untreated control cells, The bars are themean from three individual experiments. Error bars show standard errorof the mean (SEM).

FIG. 2 shows the results of a gene silencing experiment where cells ofdifferent types were transfected with siRNA using jetSI-ENDO, with andwithout PCI treatment. The results in (A) display S100A4 protein levelsin four cell lines when treated with siRNA. Grey bars show scrambledcontrol siRNA with PCI, black bars display effector siRNA without PCIand white bars display effector siRNA with PCI. Cell lines from left toright, HCT-116, SW620, OHS and RMS. The bars are the mean from threeindividual experiments. Error bars show standard error of the mean(SEM). The results in (B) show a Western blot showing different celllines from the top to the bottom, HCT-116, SW620, OHS and RMS. The upperpanel displays α-tubulin loading control and the lower panel S100A4levels. In each lower panel, lanes 1-3 show protein levels without PCItreatment: untreated control (C), scrambled control (siRNA11), andeffector (siRNA9). Lanes 4-6, show protein levels with PCI treatment:untreated control (C), scrambled control (siRNA11), and effector(siRNA9).

FIG. 3 shows results of a gene silencing experiment in OHS cells a)Dose-dependent silencing (1-5 nM siRNA) 96 hrs after irradiation b)Time-dependent silencing (24, 48 and 96 hrs) with siRNA9. Results arerepresented as percent of untreated control cells. The bars are the meanfrom three individual experiments. Error bars show standard error of themean (SEM).

FIG. 4 shows the results of gene silencing with siRNA transfected withjetSI, with and without PCI treatment 96 hrs after irradiation. Resultsshow S100A4 protein level (A) and RNA levels (B) after treating the OHScell line with 100 nM siRNA as indicated below. Black bars representsamples without PCI, and white bars represent samples subjected to PCI.Untreated control without PCI treatment was used as control for allsamples (not shown). Samples: 1 and 4) scrambled control (siRNA11), 2and 5) effector (siRNA9), 3) untreated control. The bars represent themean from three individual experiments. Error bars show standard errorof the mean (SEM).

FIG. 5 shows the distribution of fluorescence labelled siRNA in OHSfollowing transfection (200 nM) with jetSI-ENDO, with and without PCItreatment a) Delivery of siRNA without PCI treatment, from left toright: phase contrast, fluorescence and the corresponding merge, b)Delivery of siRNA with PCI treatment: from left to right: phasecontrast, fluorescence and the corresponding merge. c) Fluorescencepictures without PCI treatment displaying from left to right:fluorescence entrapped in endosomes (left hand box) and fluorescenceleakage from endosomes (right hand box), enlargement of the image withinthe left hand box, and enlargement of the image within the right handbox.

FIG. 6 shows the results of gene silencing using jetSI after treatingOHS with 100 nM siRNA and jetSI at 50% of the level recommended comparedto the standard protocol. Bars represent; 1) scrambled control siRNAwithout PCI, 2) effector siRNA without PCI, 3) untreated control withPCI, 4) scrambled control siRNA with PCI, 5) effector control siRNA withPCI. The bars represent the mean from three individual experiments.

FIG. 7 shows the structure of preferred lipopolyamines. (See also Ahmedet al, supra and Behr et al (1989) PNAS, 86, 6982-6).

FIG. 8 shows the results of gene silencing using PEI as the carrier. TheWestern blot displays a loading control (alpha-tubulin) in the upperpanel and S100A4 protein levels in the lower panel. Lane 1=untreatedcontrol (without PEI), 2=1 μl PEI+effector siRNA, 3=10 μl PEI+effectorsiRNA, 4=untreated control (without PEI), 5=1 μl PEI+effector siRNA,6=10 μl PEI+effector siRNA. Lanes 1-3 are without PCI, and lanes 4-6 arewith PCI.

FIG. 9 shows the results of gene silencing using 25 kDa PEI as thecarrier. The samples used are indicated on the Figure. A. S100A4 proteinlevel quantified by scanning of Western blots (using S100A4 siRNA withor without PCImean of 3 individual experiments where error barsrepresent SEM). B. An example of a Western blot.

FIG. 10 shows the effect of PCI on siRNA activity with different PEIcarriers used in different concentrations. S100A4 protein levels werequantified by scanning of Western blots. Black bars representtransfections without PCI, while white bars represent transfection withPCI. The results are the mean of 3 individual experiments.

FIG. 11 shows the results of experiments to determine the toxicity ofPEI alone and in combination with PCI and siRNA. The amounts of PEI andthe light doses used in the PCI experiments are indicated. A. Toxicityof PEI without PCI. The MW of the different (branched) PEI carrierstested are indicated (mean of 5 individual experiments). B. Toxicity ofthe combination of PCI, PEI (1 μg) and siRNA at different light doses.The samples tested and the light doses used are shown.Control−=untreated control (with PEI but without PCI),Scrambled−=scrambled siRNA control (with PEI but without PCI),siRNA−=S100A4 siRNA (with PEI but without PCI). Control+, Scrambled+,and siRNA+ are the same as Control−, Scrambled−, and siRNA−, but withPCI (mean of 5 individual experiments).

FIG. 12 shows PCI-induced delivery of siRNA molecules usingBeta-Cyclodextrin amine as a carrier. Lanes 1 and 4=control withoutsiRNA, lanes 2 and 5=control scrambled siRNA, lanes 3 and 6=S100A4siRNA. The tubulin control band and the S100A4 band are as shown.

FIG. 13 shows PCI-induced delivery of siRNA molecules using poly amidoamide (PAMAM) dendrimers (G2-7) with ethylenediamine core. (A) Westernblot in which upper bands represent loading control (alpha tubulin),lower bands represent S100A4 levels. The samples in the different lanesare: 1. PAMAM G6 with PCI. 2. PAMAM G7 with PCI. 3. Control with PCI. 4.PAMAM G6 without PCI. 5. PAMAM G7 without PCI. 6. Control without PCI.(B). S100A4 protein level quantified by scanning of Western blots. Blackbars represent transfections without PCI, while white bars representtransfection with PCI. The results are the mean of 3 individualexperiments. The different forms of PAMAM used are indicated on thefigure.

FIG. 14 shows the structural formula of G0, G1 and G2 PAMAM dendrimers.

FIG. 15 shows the effect of PCI on polyarginine-mediated siRNA delivery.The level of S100A4 protein was analysed by Western blotting. The upperlane represent a loading control (alpha tubulin), the lower lanerepresent S100A4 levels. The samples on the gel were as follows:C+=control with PCI, S+=control scrambled siRNA with PCI, R+=S100A4siRNA with PCI, C=control without PCI, S=control scrambled siRNA withoutPCI, R=S100A4 siRNA without PCI. 1=polyarginine MW 15,000-70,000 at 0.35μg, 2=polyarginine MW 15,000-70,000 at 0.7 μg, 3=polyarginine MW >70.000at 0.35 μg, 4 polyarginine MW >70,000 at 0.7 μg.

EXAMPLES Materials and Methods Cell Lines and Culture Conditions.

HCT-116 (colorectal adenocarcinoma) and SW620 (colorectaladenocarcinoma) were obtained from The American Type Culture Collection(Manassas, Va., USA). The OHS (osteosarcoma) and the RMS cell line wasestablished at the Norwegian Radium Hospital. All cell lines werecultured using RPMI-1640 medium (Bio Whittaker, Verviers, Belgium orGibcoBRL, Paisley, UK), without antibiotics, but supplemented with 10%fetal calf serum (FCS; PAA Laboratories, Linz, Austria) and 2 mML-glutamine (Bio Whittaker, Verviers, Belgium). Cells were grown andincubated at 37° C. in a humidified atmosphere containing 5% CO2. Allcell lines were tested and found negative for Mycoplasma infection priorto the experiments.

Light Source and Photosensitizer.

A Lumisource® (PCI Biotech AS, Oslo, Norway) was used as the lightsource. The Lumisource® is a bank of four fluorescent tubes designed toprovide homogeneous illumination of the treatment area, emitting mainlyblue light with a peak at 420 nm. The photosensitizer, disulfonatedtetraphenylporphine (TPPS_(2a)) was purchased from Porphyrin Products(Logan, Utah, USA). TPPS_(2a) was first dissolved in 0.1 M NaOH, andthereafter diluted in phosphate-buffered saline (PBS), pH 7.5, to aconcentration of 5 mg/ml and a final concentration of 0.002 M NaOH. Thephotosensitizer was light protected and stored at −20° C. until use.

siRNA Transfection without PCI.

Different transfection reagents for siRNA delivery were evaluated byusing: Lipofectin™ Reagent from Life Technologies Inc. (Gaithersburg,Md., USA), Lipofectamine™ 2000 from Invitrogen (Carlsbad, Calif., USA),FuGene 6 from Roche Diagnostics (Mannheim, Germany), siPORT™ LipidTransfection Agent from Ambion (Austin, Tex., USA), jetSI™ andjetSI™-ENDO from Polyplus transfection (Illkirch, France). Alltransfection reagents were handled according to the manufacturers'specifications. All cell lines were cultured as described in “Cell Linesand Culture Conditions”, and cultivated for 24 hrs in 6-well plates to50-70% confluence before transfection for 24, 48 or 96 hrs. Transfectionreagent alone was applied to the cells as an untreated control, inaddition to scrambled siRNA with transfection reagents.

The protocol used for jetSI/jetSI-ENDO was as follows for a standard 6well plate:

Step 1: For each well, dilute 4.2(8.4) μl of jetSI/jetSI-ENDO solutioninto 100 μl of medium. Vortex vigorously (important: do not pipet tomix) and wait for 10 min (important: do not exceed 30 minutes).Step 2: For each well, dilute 1.4 μg (100 nM) of siRNA duplex and into100 μl of medium. Vortex gently.Add the 100 μl jetSI medium solution to the 100 μl siRNA solution andmix solution at once (important: do not mix the solutions in the reverseorder)Immediately vortex-mix the solution for 10 seconds.Incubate for 30 minutes at room temperature to allow complexes to form(important: do not exceed 1 hour).During complex formation, remove the growth medium from the plates andadd 0.8 ml of fresh serum containing medium (and photosensitizer, ifused), pre-warmed at 37° C.Add the 200 μl jetSI/siRNA solution into each well and homogenize themixture by gently swirling the plate.Incubate the plate under the required cell culture conditions for 18hrs, then wash the plate three times with fresh medium and re-incubatewith 2-4 ml medium.siRNA Transfection with PCI.

Cells were cultured and transfected as in “siRNA transfection withoutPCI”, except for a few modifications. The photosensitizer TPPS_(2a) (0.5μg/ml) was added to the medium upon transfection. After 18 hrs ofincubation, cells were washed 3 times with fresh medium, and incubatedfor 4 hrs before light treatment. After 4 hrs, cells were exposed toblue light (7 mW/cm²) for different durations (60-90 sec), depending onthe cell line, and re-incubated for 24, 48 and 96 hrs before harvesting.To measure the PCI effect, effector siRNA, scrambled siRNA andtransfection reagent alone were applied in the different wells of thesame plate, with or without photosensitizer, and given the sametreatment. Cells were light protected by aluminium foil during theseexperiments.

Real-Time Reverse Transcriptase PCR of S100A4.

Total cellular RNA was isolated with the GenElute Mammalian Total RNAMiniprep Kit (Sigma-Aldrich, Steinheim, GER) and the iScript cDNAsynthesis kit (BioRad, Hercules, Calif.) was used for reversetranscription. Both kits were used according to the manufacturers'manuals. All PCRs were run in parallels, real-time detection wasobtained by use of SYBR Green I. For each PCR, 10 μl cDNA, 30 μl iQSYBRGreen Supermix (BioRad), 300 nM of each primer and nuclease freewater were added to a final volume of 60 μl. Samples of 25 μl each wasthen applied to the PCR plate. This method ensures that the parallelsare true parallels, and that there is enough PCR mix for all replicates.Primer design was accomplished using the software Primer Express fromApplied Biosystems (Applied Biosystems, Foster City, Calif.). The primerset used (forward primer 5′-AAGTTCAAGCTCAACAA GTCAGAAC-3 and reverseprimer 5′-CATCTGTCCTTTTCCCCAAGA-3′) amplifies a 79-bp segment in exon 2and 3 of the S100A4 sequence.

Real-time reactions were run on an iCycler (Bio-Rad) with the followingamplification protocol: 3 min initial denaturation at 95° C., 50 cyclesof 10 s denaturation at 95° C. and 35 s annealing/extension at 60° C.,one hold at 95° C. for 20 s followed by a hold for 1 min at 55° C., andfinally a melt curve analysis of 80 steps each for 10 s, with 0.5° C.increase until a final temperature of 95° C. The quality of the RNAsamples was verified by amplification of two housekeeping genes, TBP(forward primer 5′-GCCCGAAACGCCGAATAT-3 and reverse primer 5′-CGTGGCTCTCTTATCCTCATGA-3′) and RPLPO (forward primer 5′-CGCTGCTGAACATGCTCAAC-3′ and reverse primer 5′-TCGAACACCTGCTGGATGAC-3′). The GeneExpression Macro, version 1.1 (Biorad), was used for the quantitativecalculations. The program performs calculations based on the ΔΔ CTmethod, which allows comparison of cycle threshold values obtained usingdifferent sets of primers on the same set of samples.

Microscopy Studies.

Cells incubated with siRNA/jetSI-ENDO complexes as described (siRNAtransfection with and without PCI) and analyzed with and without PCItreatment after 48 hrs with a Zeiss inverted microscope, Axiovert 200equipped with filters for FITC (450-490 nm BP excitation filter, a 510nm FT beamsplitter, and a 515-565 nm LP emission filter), and Rhodamine(546/12 nm BP excitation filter, a 580 nm FT beamsplitter, and a 590 nmLP emission filter) Pictures were composed by the use of Carl ZeissAxioCam HR, Version 5.05.10 and AxioVision 3.1.2.1 software. Images wereprepared with Adobe Photoshop 7.0 (Adobe, San Jose, Calif.) and ZeissLSM Image Browser (Version 3).

Western Immunoblotting.

Protein lysates were prepared in 50 mM Tris-HCl (pH 7.5), containing 150mM NaCl and 0.1% NP-40 with 2 g/ml pepstatin, aprotinin (Sigma ChemicalCompany, St Louis, Mo.) and leupeptin (Roche Diagnostics, Mannheim,Germany). Total protein lysate (30 μg) from each sample was separated by12% SDS-polyacrylamide gel electrophoresis, and transferred ontoImmobilon-P membranes (Millipore, Bedford, Mass.) according to themanufacturer's manual. As a loading and transfer control, the membraneswere stained with 0.1% amidoblack. The membranes were subsequentlyincubated in 20 mM Tris-HCl (pH 7.5), containing 0.5 M NaCl and 0.25%Tween 20 (TBST) with 10% dry milk (blocking solution) before incubationwith rabbit polyclonal anti S100A4 (diluted 1:300, DAKO, Glostrup,Denmark) and mouse monoclonal anti α-tubulin (diluted 1:250, AmershamLife Science, Buckinghamshire, England) in TBST containing 5% dry milk.After washing, the immunoreactive proteins were visualized usinghorseradish peroxidase conjugated secondary antibodies (diluted 1:5000DAKO, Glostrup, Denmark), and the enhanced chemiluminescense system(Amersham Pharmacia Biotech, Buckinghamshire, England). S100A4 proteinlevels were reported as percentages of control sample and α-tubulin wasused as a loading control.

Example 1 Gene Silencing in the OHS Cell Line

OHS cells were transfected with designed to target the expression of theS100 A4 protein using various transfection systems. The standardtransfection protocol was used in each case, according to themanufacturers' instructions. When photosensitiser was used, it wasTPPS_(2a) (0.5 μg in 1 ml transfection volume, apart from jetSI where a2 ml transfection volume was used). The irradiation time in each casewas 60 seconds.

20 μM siRNA stock solution was mixed with each different transfectionreagents and transfected in 6-well plates to a final volume of 1 ml(except for jetSI and jetSI-ENDO, where the final volume was 2 ml).

siPORT Lipid=2 and 4 μl were tested, combined with 1.4 μg siRNA in 1000μl

FuGene 6=4.2 μl and 8.4 μl were tested, combined with 1.4 μg siRNA in1000 μl

Lipofectamine 2000=4.2 and 7 μl were tested, combined with 1.4 μg siRNAin 1000 μl

Lipofectin=4.2 and 7 μl were tested, combined with 1.4 μg siRNA in 1000μl

jetSI=8.4 pt, combined with 2.8 μg siRNA in 2000 μl

jetSI-ENDO=4.2 μl, combined with 1.4 μg siRNA in 2000 μl.

The siRNA molecule used was against the S100A4 mRNA sequence (GenBankaccession number NM_(—)002961). A specific siRNA having the sequence

5′-UGAGCAAGUUCAAUAAAGA-3′ 3′-ACUCGUUCAAGUUAUUUCU-5′was designed according to Elbashir et al., ((2001), Genes Dev. 15,188-200). In addition to the siRNA designed against the selected targetgene, control siRNA was designed by making a scrambled siRNA(5′-CGCAUAAGUGAAAUAGAAU-3′, 3′-GCGUAUUCACUUUAUCUUA-5′) and a BLASTsearch was performed to eliminate false hybridisation. The GC content ofthe duplexes was kept within the 30-70% range, and all siRNA moleculeswere synthesized with dTdT overhangs at their 3′ ends for optimalstability of the siRNA duplexes. siRNA molecules were ordered fromEurogentec (Seraing, Belgium).

Dried siRNA oligonucleotides were re-suspended to 100 μM in DEPC-treatedwater and stored at −20° C. Annealing was performed by separatelyaliquoting and diluting each RNA oligonucleotide to a concentration of50 μM. Then, 30 μl of each RNA oligonucleotide solution and 15 μl of 5×annealing buffer were combined, to a final concentration of 50 mM Tris,pH 7.5, 100 mM NaCl in DEPC-treated water. The solution was thenincubated for 3 min in a water bath at 95° C., followed by gradualcooling for 45 min on the workbench. Successful annealing was confirmedby 4% NuSieve agarose gel electrophoresis (data not shown).

It can be seen from FIG. 1 that PCI dependent gene silencing can beachieved through using the transfection agents jetSI and jetSI-ENDO.When these two transfection agents are used in the absence of PCI (i.e.irradiation, but in the absence of photosensitiser), gene expression isaround 75% of the control based on a measurement of protein levels usingwestern blotting however when PCI is additionally used, gene expressionis reduced to around 15% of the control.

In contrast, the use of SIPORT lipid or Fugene 6 as transfection agentsdid not achieve any significant reduction in gene expression, and theuse of lipofectamine 2000 and lipofectin achieved inhibition of geneexpression irrespective of whether PCI was also used.

Example 2 Gene Silencing in Different Cell Lines

Four different cell lines, HCT 116, SW620, OHS and RMS cells weretransfected with 50 nM siRNA (jetSI-ENDO=4.2 μl, combined with 1.4 μgsiRNA) designed to silence expression of S100A4 (see Example 1) usingthe standard jetSI-ENDO protocol, in the presence and absence ofphotosensitiser (0.5 μg/ml TPPS_(2a)). Cells were subjected toirradiation and S100A4 protein levels were determined by performingwestern blots 96 hours after irradiation (irradiation conditions were asfollows, OHS=60 sec, SW620=80 sec, HCT116=90 sec, RMS=70 sec).

Examples of the results, are shown in FIG. 2B, and the results arerepresented graphically in FIG. 2A. In each cell type, the exposure oftransfected cells to PCI treatment caused high levels of gene silencing,whereas those cells which were transfected with S100A4 siRNA but notsubjected to PCI treatment showed significantly less gene silencing. Theeffect was shown to be specific since scrambled siRNA had no effect ongene expression, either in the presence or absence of PCI. There was nosignificant difference in the gene silencing effect between thedifferent cell lines that were tested.

Example 3 Effect of siRNA Concentration and Time

The siRNA molecule designed to silence expression of the S100A4 proteinwas transfected into OHS cells at concentrations of 1 to 5 nM, using 4.2μl jetSI-ENDO, and subjected to PCI treatment as described above.

Protein levels in cell lysates were measured using western blotting andare shown as a percentage of protein levels in untreated control cellsin FIG. 3A.

The gene silencing effect increased with the concentration of siRNA towhich the cells were exposed, although there is little differencebetween the silencing effects seen with 4 nM and 5 nM siRNA.

The scrambled siRNA molecule was also used in all experiments and wasshown not to affect gene expression. As such, the gene inhibition effectis specific.

FIG. 2B shows the effect of time on gene silencing by altering theperiod of time between irradiation of the cells and harvesting of celllysates for analysis. It can be seen that the longer the cells are leftafter irradiation and before harvesting, the more inhibition of geneexpression is observed.

Example 4 Gene Silencing in OHS Cells after siRNA PCI Treatment

OHS cells were transfected with 100 nM siRNA or scrambled siRNA asdescribed above and the effect of PCI treatment on protein and mRNAlevels were determined 96 h after irradiation by western blotting andRT-PCR and compared to protein and mRNA levels in untreated controls.

The results shown in FIG. 4 indicate that, with respect to both proteinand mRNA levels, large amounts of gene silencing of the S100A4 gene isobserved when the siRNA specific for this gene is transfected with jetSIENDO and the cells are subjected to PCI treatment (lane 5 of FIGS. 3Aand B). A small amount of reduction in protein and RNA levels is seenwithout PCI treatment (lane 2 of FIGS. 3A and B), but this is markedlyenhanced by the PCI treatment.

Example 5 Cytosolic Delivery of Labelled siRNA

OHS cells were transfected with 200 nM FITC labelled siRNA using jetSIENDO ((jetSI-ENDO=8.4 μl, combined with 2.8 μg siRNA in a 1000 μltransfection volume=200 nM siRNA solution), with and withoutphotosensitiser. Transfected cells were then subjected to irradiation.

The cells were inspected using phase contrast microscopy andfluorescence microscopy. By comparing the images, it was seen that inthe absence of PCI treatment, the siRNA remains in a punctatedistribution, which is representative of a distribution in endocyticvesicles (FIGS. 5 a and c). There is also some leakage seen (right handbox in FIG. 5 c). In contrast, following PCI treatment the labelledsiRNA is seen to be distributed throughout the cytoplasm and in thenucleus (see FIG. 5 b).

This demonstrates that PCI treatment is required for siRNA to bedelivered to the required compartment of the cell, and that the deliveryis dependent on PCI treatment.

Example 6 Gene Silencing Using siRNA-PCI Requires the Use of LessCarrier than Standard Transfection

OHS cells were transfected with 100 nM siRNA using jetSI. In contrast tothe experiments described above, jetSI was used at a lowerconcentration, i.e. at 50% of that recommended in the standard protocol.The standard protocol for a 6-well plate is: 2.8 μg siRNA+8.4 μl jetSIin 2000 μl medium, resulting in 100 nM siRNA in each well.

In contrast, for a 6-well plate, 1.4 μg siRNA was mixed with 4.2 μljetSI in 1000 μl medium, resulting in 100 nM siRNA in each well. Thetotal amount of complexes was thus reduced by 50%.

Following transfection, cells were either subjected to PCI treatment(the light dose for this experiment was 30 sec with 0.5 μg/ml TPPS2a) orleft untreated, and gene silencing was measured using RT-PCR. Theeffector siRNA was able to reduce expression of the S100A4 gene to lessthan 20% of the untreated control, whereas little reduction was seenwithout PCI, and with the use of scrambled siRNA (either with or withoutPCI treatment).

This demonstrates that not only does the combined use of carriers withPCI offer the advantage of selective release of the siRNA molecule, butthat high levels of gene silencing can be achieved using lowerconcentrations of the transfection agents, when combined with PCI. Bycomparison of FIG. 6 lane 5 with FIG. 1 lane 5 it can be seen that evenusing only 50% of the transfection agent that is used in Example 1, whenPCI is also used, the degree of gene inhibition is much greater.

Example 7 Transfection Using PEI as Carrier

The siRNA target was selected against the S100A4 mRNA sequence (GeneBank accession number NM_(—)002961). siRNA 481-499 was used as effector(for sequence see Example 1).

Polyethylenimine (PEI) was evaluated for PCI-induced delivery. Celllines were cultured (RPMI-1640 supplemented with 10% FBS, 10 mlL-glutamate, 10 ml Hepes) in 6-wells plates to 50-80% confluence beforetransfection. 100 nM siRNA was used as the standard concentration.

The PEI used was from Sigma and was diluted in sterile water and a stocksolution containing 1000 μl PEI and 9000 μl sterile water was made. Fromthe stock solution 1 and 10 μl was used to transfect of cells with 1.4μg siRNA (100 nM in each well).

PEI from Sigma was used (408719 Polyethylenimine (average Mw˜800 by LS,average Mn˜600 by GPC, branched, Low molecular weight, water-free)).

For the transfection, two solutions were made up, solution A, siRNA wasdiluted in 100 μl of serum free (OPTI-MEM I) medium. Solution B: PEI wasdiluted in 100 μl of serum free medium. Solutions A and B were mixed bygently mixing and incubated in room temperature for 30 min. The mixedsolution was then added to the cells (1 ml of 100 mM siRNA).

PCI treatment was performed as described above for jetSI. Light dosesfor the PEI experiment was 40 sec. Protein levels were measured byWestern blotting 96 hrs after irradiation.

It can be seen from FIG. 8 that S100A4 protein expression is reduced inthe samples shown in lanes 5 and 6, i.e. the samples which have beensubjected to transfection with PEI, and PCI treatment.

Example 8 Effect of PCI on siRNA Activity Using a 25 kDa PEI Carrier

siRNA transfection. All cell lines were cultured as described in “CellLines and Culture Conditions”, and plated in 6-well plates at 25-50%confluence before transfection. siRNA and carrier were complexed bygentle mixing and incubated for 30 min before being added to cells.Cells were transfected with siRNA, carrier, and either with or withoutphotosensitiser (TPPS_(2a)=0.5 μg/ml) and incubated for 18 hrs, thenwashed 3 times with fresh medium and re-incubated for 4 hrs before lighttreatment. After 4 hrs, cells were exposed to blue light (7 mW/cm²) fordifferent durations (0-60 s), depending on the experiment, andre-incubated for 96 hrs before harvesting. To measure the effect of PCIupon gene silencing, specific siRNA, scrambled siRNA, and transfectionreagent alone were applied in the different wells of the same plate,with or without photosensitiser, and given the exact same treatment.Cells were light protected by aluminum foil during the experiments.

siRNA mediated S100A4 gene silencing was measured at the protein levelwith and without PCI treatment using branched 25 kDa polyethylenimine(PEI) (1 μg/ml) and an siRNA targeting the S100A4 gene at a 100 nMconcentration. The protocol described above was followed, using a lightdose of 30 s, with both complexation and transfection in serumcontaining medium. FIG. 9A shows that with the use of PCI S100A4 siRNAtypically reduced the S100A4 level down to 5-15% of the level inuntreated controls (treated with PEI but without siRNA). In contrast,S100A4 siRNA without PCI was only able to reduce the S100A4 level downto 100-80% of the control. Untreated control levels were comparable tocontrols where a scrambled siRNA was used instead of the S100A4 specificsiRNA (data not shown). In FIG. 9B, the experiment is represented byWestern Blots. The upper bands represent a loading control (alphatubulin) and the lower band represents S100A4 levels, as indicated onthe figure. As can been seen from the blots, S100A4 is significantsilenced with siRNA when using PCI, as compared to the situation forcells receiving S100A4 siRNA that were not treated with PCI, where nosignificant gene silencing could be detected.

Example 9 Effect of PCI on siRNA Activity with Different PEI CarriersUsed at Different Concentrations

PCI effects on of S100A4 siRNA activity was investigated for usingvarious branched PEI formulations at different concentrations (0.1μg/ml, 1 μg/ml, 10 μg/ml and 100 μg/ml, 1 ml medium was used per well).The PEI species tested (all branched) were as follows:

PEI MW (Da) 1 800 2 1200 3 1300 4 1800 5 2000 6 25000

The protocol described in Materials and Methods was followed, using alight dose of 30 s, with both complexation and transfection in serumcontaining medium. The siRNA was used at a concentration of 100 nM. Ascan be seen from FIG. 10 the use of PCI can significantly enhance thegene silencing effect of the S100A4 siRNA with different PEI carriers.This effect is especially prominent with lower amounts of PEI (1 and 10μl/ml) where the effect without PCI was very modest, except for PEI 4(MW 1800) at 10 μg/ml. At these two PEI concentrations PCI significantlyenhanced the gene silencing effect of all the tested PEI species, exceptfor PEI 1 (MW 800).

Without PCI the degree of gene silencing increases with increasing PEIconcentration, with PCI this effect is not so pronounced, except that nogene silencing (neither with nor without PCI) can be observed with 0.1μg/ml PEI. One possible explanation for this is that this amount of PEIis not high enough to complex all the siRNA, leading to negativelycharged complexes that are not taken up by the cells. Without PCI thereseems to be a trend towards increased gene silencing with increasingmolecular weight of the PEI carrier; with PCI this effect is not soconspicuous, again with exception of PEI 1 (MW 800). The effect of PEIwithout PCI at higher MWs and higher amounts is probably due to thereported endosomolytic properties of PEI working only at highconcentrations of PEI.

This shows that PCI can substitute this effect making it possible to uselow amounts and low MWs of polyethylenimine, a property that is veryadvantageous to avoid toxicity and other concerns with PEI carriers.

Example 10 Toxicity Studies

The toxicity of different PEI formulations (MW 800-25.000) alone(without PCI treatment) was first assessed. In this assay, OHS cellswere plated in 96-well plates and allowed to adhere overnight in serumcontaining medium. The medium was then discarded, and cells wereincubated with medium and various PEI formulations under differentconcentrations for 20 hrs. The PEI containing medium was then discarded,and MTS solution (from Promega, Madison, Wis., USA) was added to eachwell (1:6 dilution, 100 μl/well), and plates were re-incubated foranother 4 hrs. Absorbance at 490 nm was measured.

As can be observed from FIG. 11A, toxicity increased with increasingmolecular weight of the PEI formulation (e.g. 25000 PEI vs. 800 PEI) andwith increasing amounts of PEI. Importantly, PEI formulations at 1 μg/ml(not shown) and 10 μg/ml did not show significant toxicity. For thesesamples, a significant biological effect of the siRNA could be achievedwith PCI, while the effect without PCI was very modest (see Example 9).

The toxicity of PEI with siRNA/PCI treatment was also assessed. OHScells were plated in 6-well plates and allowed to adhere overnight inserum containing medium. The medium was then discarded, and cells in6-well plates were transfected with PEI alone, scrambled siRNA andspecific siRNA, with or without photosensitiser. Cells were thenincubated overnight and subsequently washed and treated with blue light(PCI) as described in “siRNA transfection”. After re-incubation for 44hrs, 166.6 μl MTS solution) was added to each well and plates werere-incubated for another 4 hrs. Absorbance at 490 nm was measured. FIG.11B shows the toxicity of the combined treatment with PEI (MW 25 000) at1 μg/ml, 100 nM siRNA and PCI at different light-doses. As can be seenfrom the graph, the PCI treatment did not induce significant toxicity atthe light-dose used (30 s) in all the PEI gene silencing experiments,which is a light-dose at which a strong gene silencing effect wasinduced by PCI (see FIG. 10). Even at the higher light dose of 40 s nocytotoxicity of the PCI treatment could be observed, showing that PCIcan induce fully enhance siRNA delivery without giving cytotoxiceffects.

Example 11 PCI-Induced Delivery of siRNA Molecules Using β-CyclodextrinAmine as a Carrier

The effect of PCI on Beta-cyclodextrin amine mediated siRNA delivery wasinvestigated. A light-dose of 60 s was used in these experiments, andthe protocol described in Materials and Methods was followed.Beta-cyclodextrin amine as described above with n=6 and X=4 was dilutedin sterile water and complexation and transfections were performed inserum-free medium. As can been seen from the Western blot (FIG. 12)Beta-cyclodextrin amine at 100 μg/ml (1 ml was used per well) complexedwith 50 nM (0.7 μg) siRNA was effective for PCI induced siRNA delivery(lane 3), while under these conditions delivery without PCI wasineffective (lane 6). The Beta-cyclodextrin amine used in this studyconsists of Beta-cyclodextrin molecules conjugated through an aminebridge responsible for binding to siRNA, and is described in Hwang S. J.et al. (2001, Bioconjugate Chem. 12, 280-290).

Example 12 PCI-Induced Delivery of siRNA Molecules Using Poly AmidoAmide (PAMAM) Dendrimers (G2-7) with Ethylenediamine Core

Poly amido amide (PAMAM) dendrimers were evaluated for PCI-induced siRNAdelivery. PAMAM was diluted in sterile water and transfections wereperformed in serum containing medium. Different types of PAMAMdendrimers (G2-7) at 100 μg/ml (1 ml was used per well) were complexedwith 100 nM (1.4 μg) siRNA and transfection was carried out according tothe procedures described above. A light-dose of 30 s was used in theseexperiments. As can be seen from the Western blot (FIG. 13A) PCI couldstrongly enhance the activity of siRNA (lanes 1 and 2) under conditionswhere siRNA/PAMAM alone was ineffective for gene silencing (lanes 4 and5). As can be seen in FIG. 13B this effect was apparent also for severalother types of PAMAM dendrimers, indicating that PCI can generallyenhance siRNA delivery by polyamine-based dendrimers.

The different types of PAMAM used in the study containing differentamounts of surface amine groups are as follows:

G2=Molecular Formula: [NH₂(CH₂)₂NH₂]:(G=2); dendri PAMAM(NH₂)₁₆G3=Molecular Formula: [NH₂(CH₂)₂NH₂]:(G=3); dendri PAMAM(NH₂)₃₂G4=Molecular Formula: [NH₂(CH₂)₂NH₂]:(G=4); dendri PAMAM(NH₂)₆₄G5=Molecular Formula: [NH₂(CH₂)₂NH₂]:(G=5); dendri PAMAM(NH₂)₁₂₈G6=Molecular Formula: [NH₂(CH₂)₂NH₂]:(G=6); dendri PAMAM(NH₂)₂₅₆G7=Molecular Formula: [NH₂(CH₂)₂NH₂]:(G=7); dendri PAMAM(NH₂)₅₁₂

Generation Measured Surface amine (G) MW diameter (nm) groups 2 3,256 2916 3 6,909 36 32 4 14,215 45 64 5 28,826 54 128 6 58,048 67 256 7116,493 81 512

Example 13 PCI-Induced Delivery of siRNA Using Poly-L-ArginineHydrochloride as the Carrier

Polyarginine was diluted in sterile water and transfections wereperformed in serum free medium. Two types of polyarginine carriers(molecular weight of 15000-70000 and >70000) were tested. Polyarginineat 0.35 or 0.7 μg/ml (1 ml was used per well) were complexed with 100 nM(1.4 μg) siRNA, and the protocol described above was followed, assessingS1004A expression by Western blotting after giving a light-dose of 30 s.As can be seen from FIG. 15, there is a significant difference in genesilencing efficacy between PCI-treated and non-treated samples. Thus,while all the PCI-treated samples (R+—samples in FIG. 15) showssignificant gene silencing, no silencing effect can be observed incorresponding samples not treated by PCI (R-samples in FIG. 15). Thus,PCI is effective in inducing gene silencing with both the differentpolyarginine carriers tested and at both the concentrations used,showing that PCI can significantly enhance siRNA delivery by a cationicpeptide-based carrier.

1. A method for introducing an siRNA molecule into the cytosol of acell, said method comprising i) contacting said cell with an siRNAmolecule, a carrier and a photosensitising agent, and ii) irradiatingthe cell with light of a wavelength effective to activate thephotosensitising agent, wherein said carrier comprises a cationicpolyamine selected from (a) a lipopolyamine in a non-liposomalformulation, (b) polyethyleneimine (PEI) having an M_(n) value of500-20000 by GPC, (c) a betacyclodextrin amine polymer of formula

wherein n is an integer from 1 to 1 inclusive and n is an integer from 4to 10 inclusive, (d) an amide group containing dendrimer, and (e) acationic peptide.
 2. The method of claim 1 wherein the lipopolyaminecontains primary or secondary amine groups, or a mixture thereof.
 3. Themethod of claim 1, wherein the polyamine region of the lipopolyamine hasat least 2 nitrogen atoms and a charge of at least +1 at physiologicalpH.
 4. The method of claim 1 wherein at least one nitrogen containinggroup of said carrier is uncharged at physiological pH.
 5. The method ofclaim 4 wherein the pKa at which the last amine of the lipopolyamine isprotonated on decreasing pH is greater than or equal to 5.5.
 6. Themethod of claim 1 wherein the polyamine region of the lipopolyamine hasthe formula (I)

in which m is an integer greater than or equal to 2 and n is an integergreater than or equal to 1, wherein m may be the same or different whereit appears in said formula, at each position R¹ is a hydrogen or alinking group to the lipo portion of the lipopolyamine, or the lipoportion itself, and may be the same of different at each carbon atom, R²is a hydrogen or a linking group to the lipo portion of thelipopolyamine or the lipo portion itself, wherein only one of R¹ and R²is a linking group to the lipo portion of the lipopolyamine, or the lipoportion itself and wherein when R¹ or R² is the linking group attachedto said lipo portion or the lipo portion itself, formula (I) is saidlipopolyamine.
 7. The method of claim 6 wherein m is between 2 and 6inclusive and n is between 1 and
 5. 8. The method of claim 7 wherein nis
 3. 9. The method of claim 6 wherein R² is H.
 10. The method of claim6 wherein the polyamine region is represented by the following formula

Wherein R^(1a) to R^(1j) are as R¹ defined in claim
 7. 11. The method ofclaim 10 wherein R^(1a) is a linker and the remaining R¹ groups arehydrogen.
 12. The method of claim 6 wherein R¹ or R² represent ahydrogen atom or a radical of general formula II:

in which R³ and R⁴, which may be identical or different, each representa saturated aliphatic radical C_(p) H_(2p+2) or unsaturated aliphaticradical C_(p) H_(2p) or C_(p) H_(2p−2), p being an integer between 12and 22 inclusive, and R⁵ represents a hydrogen atom or an alkyl radicalcontaining 1 to 4 carbon atoms optionally substituted with a phenylradical, with the proviso that only one of R¹ and R² can represent aradical of general formula (II).
 13. The method of claim 6 wherein R¹and R² each represent a hydrogen atom or a radical of general formulaIII:

in which X represents a methylene group (—CH₂—) or a carbonyl group(—CO—), and R⁶ and R⁷, which may be identical or different, eachrepresent a saturated aliphatic radical C_(p′) H_(2p′+2) or unsaturatedaliphatic radical C_(p′) H_(2p′) or C_(p′) H_(2p′−2), and p′ is aninteger between 11 and 21 inclusive, with the proviso that only one ofR¹ and R² can represent a radical of general formula (III).
 14. Themethod of claim 6 wherein n is an integer between 2 and 5 inclusive, andthe values of m in the different fragments

are identical.
 15. The method of claim 6 wherein n is between 2 and 5inclusive, and the values of m in the different fragments

are different.
 16. The method of claim 6 wherein n is equal to 3 and thevalues of m in the fragments

are identical or different and represent 3 or 4, and either R¹ or R²represents: (i) a radical of general formula (II) in which R³ and R⁴each represent an alkyl radical containing 12 to 22 carbon atoms and R⁵represents a hydrogen atom, or (ii) a radical of general formula (III)in which R⁶—X— and R⁷—X— each represent an alkanoyl radical containing12 to 22 carbon atoms.
 17. The method of claim 1 wherein the lipophilicregion of said lipopolyamine is any saturated or unsaturated hydrocarbonchain, cholesterol or other steroid, a natural lipid or a syntheticlipid capable of forming lamellar or hexagonal phases.
 18. The method ofclaim 17 wherein the length of the hydrocarbon chain is from 10 to 30carbons in length.
 19. The method of claim 1 wherein the lipopolyamineis selected from 5-carboxyspermylglycinedioctadecylamide (DOGS),dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES),2,3-dioleyl-oxy-N[2-sperminecarboxyyl-amido]ethyl-N,N-dimethyl-1-propanaminiumtrifluoracetate (DOS PA), 1,3-dioleoyloxy-2-(6-carboxyspermine) (DOSPER)and RPR-120535.
 20. The method of claim 1 wherein the lipopolyamine isselected from 5-carboxyspermylglycinedioctadecylamide (DOGS), anddipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES). 21.The method of claim 1 wherein the carrier is JetSI™ or JetSI-ENDO™, orTransfectam®.
 22. The method of claim 1 wherein said carrier molecule isa branched PEI molecule.
 23. The method of claim 22, wherein themolecular weight of the PEI is less than 50 kDa, preferably less than 25kDa.
 24. The method of claim 1 wherein said carrier molecule is a PAMAMdendrimer molecule, preferably a generation 2-6 PAMAM dendrimermolecule.
 25. The method of claim 1 wherein said carrier molecule isPoly-L-Lysine, Poly-D-Lysine, Poly-Histidine, Histidylated poly-lysineand Poly-ornithine or a copolymer of L or D lysine, L or D arginine, Lor D histidine and/or ornithine residues with one or more other aminoacids.
 26. The method of claim 1 wherein the siRNA molecule is 12-28nucleotides long.
 27. The method of claim 1 wherein said cell is amammalian cell.
 28. The method of claim 1 wherein the photosensitisingagent is selected from a porphyrin, phthalocyanine, purpurin, chlorin,benzoporphyrin, lysomotropic weak base, naphthalocyanine, cationic dye,tetracycline or a derivative thereof, 5-aminolevulinic acid and/oresters thereof, preferably TPPS₄, TPPS_(2a), AlPcS_(2a), TPCS_(2a),5-aminolevulinic acid or esters of 5-aminolevulinic acids orpharmaceutically acceptable salts thereof.
 29. The method of claim 1,further comprising the additional step of contacting said siRNA withsaid carrier.
 30. The method of claim 1 wherein the siRNA molecule andthe carrier molecule are contacted with one another for 20-40 minutesbefore being contacted with the cell.
 31. The method of claim 1 wherein10 nM-200 nM siRNA is used for transfection.
 32. The method of claim 1wherein a photosensitizer carrier selected from a polycation,polyethyleneimine, a dendrimer, a cationic lipid and a peptide isadditionally present.
 33. The method of claim 1 wherein the siRNA ismixed with the carrier so as to form a complex, which is thenadministered to the cell simultaneously or sequentially with thephotosensitising agent.
 34. The method of claim 1 wherein said method isperformed by contacting said cell with a photosensitising agent,contacting said cell with the carrier and the siRNA molecule to beintroduced and irradiating said cell with light of a wavelengtheffective to activate the photosensitising agent, wherein saidirradiation is performed prior to the cellular uptake of said siRNAmolecule and said carrier into an intracellular compartment containingsaid photosensitising agent, preferably prior to cellular uptake of saidmolecule and said carrier into any intracellular compartment.
 35. Amethod of inhibiting the expression of a target gene by introducing ansiRNA molecule into a cell containing said target gene by a method asdefined in claim 1 wherein said siRNA molecule specifically inhibitsexpression of said target gene.
 36. A cell or a population of cellscontaining an siRNA molecule which has been internalised into thecytosol of said cell, which cell is obtainable by a method of claim 1.37. A composition containing an siRNA molecule and a carrier molecule asdefined in claim 1, and optionally separately also a photosensitizingagent as defined in claim
 1. 38. The composition of claim 37, containinga photosensitizing agent.
 39. A composition comprising the cell or apopulation of cells according to claim
 36. 40. The composition of claim37 for use in therapy.
 41. A kit comprising an siRNA molecule, a carriermolecule and optionally a photosensitizing agent as defined in claim 1.42. (canceled)
 43. (canceled)
 44. (canceled)
 45. A method of treating orpreventing a disease, disorder or infection in a patient comprisingintroducing an siRNA molecule and carrier into one or more cells invitro, in vivo or ex vivo according to the methods as defined in claim35 and where necessary administering said cells to said patient.
 46. Amethod of treating or preventing a disease, disorder or infection in apatient comprising introducing the cell or a population of cells asdefined in claim 36 to said patient.
 47. The method of claim 45 which isused to treat a disease which is typified by abnormal gene expression orwhich would benefit from suppression of one or more genes, preferablycancer.